text:
The Cinematic Tango: Contemporary Argentine Film
By Tamara Falicov
El Abrazo Partido (Lost Embrace), 2004
Argentine Cinema Makes a Comeback
Throughout the roughly one hundred years of its existence, Argentine cinema has had to compete against the Hollywood behemoth to garner an audience. Sadly, it was largely successful in only two periods of its film history: during the 1930s–50s, when the studio system produced high-quality, popular genre films, and once again – at least four decades later – in the early 1990s, when multimedia conglomerates began investing in blockbuster films that brought back audiences in droves.
During the post-dictatorship period beginning in 1983 when President Alfonsín was democratically elected to office, national cinema was utilized by the state as a public relations tool abroad. The majority of films produced presented tales of the recent horrible past, and provided testimony to the openness and renewal of Argentine culture in a time free of political censorship and violent repression. The Radical Party (UCR), a moderate political party, considered the cinema a bastion of high culture, and films were exported to scores of international film festivals. During this time films were mainly shown in art houses and were made in a similar vein to the European art house aesthetic. Although the films were successes in middle – to upper-middle-class film festival crowds, they were not large financial successes at the box office, and many working class Argentines did not pay to see them.
Although Alfonsín's government looked to the European model for cinema aesthetics, for President Menem (1989-1999) the model was the U.S. Hollywood film industry – higher-budget movies with special effects and blockbuster marketing techniques. Menem, taking a more Peronist view of mass popular culture, considered films to be fashioned for a domestic audience. His two administrations were marked not by an interest in promoting Argentina's image at prestigious film festivals as much as by an active attempt to cultivate the national cinema's industrial capabilities. Under Menem films were made with a glossy, globalised aesthetic and appealed to a wide general audience, thus boosting the economic viability of the industry. Films such as buddy movie Comodines (Cops, 1997) thriller La furia (The Fury, 1997), and others were imitative of Hollywood fare, but increased admissions to movie theatres. However, state support for a national culture used to inspire, educate, and cultivate a national sense of community was compromised.
El Cielito, 2004
Beginning in the mid-1990s, young directors, the majority of whom had graduated from one of many film schools in Argentina, began producing low-budget, independent films in a style that earned this group the classification of the New Independent Argentine Cinema. Part of this upsurge had to do with a small grants program that was initiated by the National Film Institute (INCAA) in the mid-1990s. These recent graduates have made short films (cortometrajes), and then have gone on to raise funds through co-production funding (Hubert Bals Fund at the Rotterdam film festival, the Fond Sud program from France among others). They have relied on their own networks of like-minded young people rather than depend on the traditional film sector structure (the film union, established director's associations, and the few film studios still in existence). This cinema is different from the previous auteurs of Argentine cinema: some directors have created gritty, realist dramas in a aesthetic vein reminiscent of the political cinema in of the New Latin American Cinema movement of the 1960s and 1970s (symbolized by the Argentine film La hora de los hornos (Hour of the Furnaces) (1968)), but rather than create overtly polemical statements or march under the banner of a political movement, they are working to expand the notion of Argentine citizenship to include subjects and characters who have traditionally been invisible or excluded from Argentine screens. Examples are the Bolivian immigrant in Adrián Caetano's Bolivia (2000); the young girl protagonist in Lucrecia Martel's La niña santa (The Holy Girl, 2004); lower middle-class Jewish characters such as Ariel's family in Daniel Burman's El abrazo partido (Lost Embrace, 2004); the Korean-Argentine characters in the film Do U Cry 4 Me Argentina? (2005) directed by Bae Youn-suk; Laura, a bisexual video editor who is Ariel's love interest in Esperando al mesías (Waiting for the Messiah, 2000), and Albertina Carri's Los Rubios (The Blonds, 2003). Carri's film is an experimental narrative in which the actor who plays the role of Carri is challenging the mythos and memories of her "disappeared" parents during the Dirty War.
These filmmakers want to break with the notion of an Argentine exceptionalism, whereby Argentina is viewed as different from other Latin American countries due to its large immigration from Italy and Spain (i.e., European background and aspiration) and distancing from an indigenous past. In other words, this younger generation of filmmakers in large part does not identify with a European-influenced and -inflected culture. Rather, these young filmmakers identify with ethnic minorities and working-class people in Argentina and project a more varied and heterogeneous face of national identity in Argentina. They show films from a grittier, working-class perspective (especially after the economic crisis of 2001), as shown by such films as Un oso rojo (A Red Bear, 2002) by Adrian Caetano, El bonarense (2002) by Pablo Trapero, and Taxi, un encuentro (Taxi, an Encounter, 2001) by Gabriela David. Additionally, there are films that work to deconstruct or disrupt the hegemony of the middle- or upper-middle-class family, as in La cienága (The Swamp, 2001), Cama Adentro (Live-In Maid) (2004).
Rapado (Skinhead), 1992
It is these filmmakers who will continue to push the boundaries of Argentine national identity through a low-budget cinema. Since their auspicious debut in the mid-1990s, however, some of these first time directors such as Pablo Trapero and Adrián Caetano, are now so esteemed and well recognised that each were recently funded by larger production companies for their next projects. Trapero, in addition to starting his own production company, Matanza cine, collaborated with the producers of Comodines, Pol-Ka, (who partner with Disney) on his feature, Familia Rodante (Rolling Family, 2004). Adrian Caetano worked with celebrity television host Marcelo Tinelli's company to produce an eleven-part television series, Disputas (Catfight) before going to direct and co-write Crónica de una fuga (Buenos Aires, 1977) which received production funds and distribution rights by 20th Century Fox-Argentina. Although some filmmakers are moving up the ranks and gaining more commercial exposure (as witnessed by long time low-budget auteur Alejandro Agresti's recent film Valentín (2002) which was distributed in the U.S. by Miramax, and his first Hollywood film for Warner Brothers, The Lake House (2006) starring Keanu Reeves and Sandra Bullock, (a Korean remake of the film Il Mare (2000)), these still are the exceptions rather than the rule. This handful of "crossover" directors calls the term "independent" into question, and this was hotly debated at the Buenos Aires International Independent Film Festival in April 2004. Despite the fact that there was no resolution, it calls into question the idea that Argentina has a full fledged film industry or not. The larger studios would claim that there isn't, thus affording themselves state subsidies, and other, smaller director producers would say that there is.
One issue is clear: the New Argentine cinema is winning accolades abroad, witnessed by recent awards at Cannes, Berlin, and San Sebastian: Daniel Burman's El abrazo partido won the Silver Bear prize at the Berlin Film Festival in 2004 (Grand Jury Prize), María Victoria Menis' El cielito (Little Sky) (2004) won multiple critics' awards at the San Sebastian Film Festival, and Paz Encina's Hamaca Paraguaya (Paraguayan Hammock, 2006) won a Critic's Prize in the Un Certain Regard category at the Cannes Film Festival, all films made by newer directors (many of them women, the number which is unprecedented) which captured the international scene. The irony, of course, is that few Argentines have come to see these films. Figures for these critically acclaimed films range on average from $100,000-250,000 and producers today claim that a medium budget film (to a tune of 1.5 million dollars) film must make $500,000 to turn a profit, since ticket prices are so low. Thus, while Argentina is making a comeback on the international film festival scene, there are still questions about how to keep home audiences motivated to see national films. These tensions grapple many national film industries, but Argentina's rich cinematic history attests to Argentina's attempts at remaking and renewing its cinematic art and industry.
Tamara L. Falicov teaches Latin American cinema studies at the University of Kansas. This extract is taken from her forthcoming book, The Cinematic Tango: Contemporary Argentine Film (Wallflower Press, 2007).
Vertigo Volume 3 | Issue 4 | Winter 2007


The Jewish people received the Torah on Mt. Sinai and were ready to enter the land of Israel. There was a consensus of opinion amongst the people that we should send spies to see if it was feasible to conquer the Land. Moshe knew that the Almighty's promise to give the Land included a guarantee to conquer it. However, one of the principles of life which we learn from this portion is: the Almighty allows each of us the free will to go in the direction we choose. Even though one man and the Almighty is a majority, Moshe — by Divine decree — sent out the princes of the tribes (men of the highest caliber) to spy out the land.
Twelve spies were sent. Ten came back with a report of strong fortifications and giants; they rallied the people against going up to the Land. Joshua ben Nun and Calev ben Yefunah (Moshe's brother-in-law) tried to stem the rebellion, but did not succeed. The Almighty decreed 40 years of wandering in the desert, one year for each day they spied in the land of Israel. This happened on the 9th of Av, a date noted throughout Jewish history for tragedy — the destruction of both Temples in Jerusalem and the expulsion of the Jews from Spain amongst them.
What is our lesson? We often see situations and come to erroneous conclusions. We must be very careful because often times there are factors that we are unaware of or don't take into consideration. It is incumbent upon us to judge people favorably … unless we're very sure.


text:
"Money is something that you can reinvest in your business"
Natalya Kaspersky
IT industryEntrepreneurshipRetail tradeDigitalization
World Economic Forum Annual Meeting 2019 Overview
Globalization/regionalizationEvents4th Industrial Revolution
Vladimir Medinskiy
Aide to the President of the Russian Federation; Chairman, Russian Military Historical Society
Victoria Abramchenko
Dmitry Medvedev
Deputy Chairman of the Security Council of the Russian Federation
Alexey Miller
Chairman of the Management Committee, Deputy Chairman of the Board of Directors, Gazprom
Mikhail Zadornov
President – Chairman of the Management Board, Otkritie FC Bank
Igor Shuvalov
Chairman, VEB.RF
Darren W/. Woods
Chief Executive Officer, Exxon Mobil Corporation
President of the French Republic
Yury Sentyurin
Secretary General, Gas Exporting Countries Forum (GECF)
Adapting to Climate Change: Challenges and Opportunities
KEY CONCLUSIONS
Global economy has set a course for decarbonization
Decarbonization of the global economy cannot be avoided. The challenges we will have to face are enormous and we must address them quickly — Selwin Charles Hart, Special Adviser to the Secretary-General on Climate Action, United Nations.
There is an ongoing change in the attitudes of society and companies. <…> Today the demand for the environmental agenda is higher than before. Among other things, last year's crisis led us to this and gave us an additional impetus to discuss this agenda. A person now no longer just buys a product, they want to know what impact the process of its production has had on the environment — Alexander Novak, Deputy Prime Minister of the Russian Federation.
Compared to 1990, our country has reduced greenhouse gas emissions. <…> We continue environmentally oriented modernization of our industries — Victoria Abramchenko, Deputy Prime Minister of the Russian Federation.
The population of the countries that ignore these initiatives has no future — Enric Sala, Explorer in Residence, National Geographic Society.
Climate agenda as a tool of economic competition
The climate does not have political views. It would be better to consider the climate outside of relations associated with trade wars, for example — Victoria Abramchenko, Deputy Prime Minister of the Russian Federation.
The efficiency of the implementation of climate projects in Russia is tens of times higher than anywhere else. <...> It is important that the efforts that we have made are recognized and counted in relation to export — Maksim Reshetnikov, Minister of Economic Development of the Russian Federation.
Many countries have announced that they will achieve carbon neutrality by 2050, but in reality, emissions are increasing. It is not enough to show ambition, you need to act. <…> The Russian Federation is consistent in its actions; we will not call for anything that is groundless. First you need to carry out research work. It is only after this that will we call for carbon neutrality — Ruslan Edelgeriyev, Advisor to the President of the Russian Federation; Special Presidential Representative on Climate Issues.
World economy is not ready for dramatic environmental reforms
Energy transition is inevitable. There are different options for its speed though. <…> According to the International Energy Agency, we need to completely stop financing the oil and gas industry starting tomorrow. Everyone understands that this is unrealistic. <...> Around 30 countries have announced that they will give up the coal power. All this sounds politically beautiful ... but there are a lot of questions that are not answered. For example, a UN representative said that one of the issues of the fight against the coal industry would be discussed in Glasgow. <...> I do not and will not understand why exactly such goals are set and what workers in Kuzbass, for example, should do with these goal — Alexander Novak, Deputy Prime Minister of the Russian Federation.
We understand how all this will be done, by what means, changing what elements of business systems — Dmitry Konov, Head of the Administration of the Gusevsky City District Municipal Structure.
Closing carbon markets has its disadvantages. <…> We see discrimination and double standards. They do not lead to a global reduction in greenhouse gas emissions. On the one hand, they call for national ambition, but at the same time they close the markets — Ruslan Edelgeriyev, Advisor to the President of the Russian Federation; Special Presidential Representative on Climate Issues.
Use the potential to tackle environmental challenges
To prevent climate change and disasters, we must significantly reduce emissions and innovate technologies. This is especially true for pollution with carbonaceous substances — Enric Sala, Explorer in Residence, National Geographic Society.
In the Arctic zone there is permafrost, and we see large-scale floods and freezing rains. <…> We are now creating a system of carbon polygons – sites in different natural climatic zones. Currently there are already eight of them. <…> We are launching a pilot project in Sakhalin Region to work out the mechanisms. By 2025, we plan that it will become carbon neutral. This year we must finish working on adaptation plans for the most vulnerable sectors of the economy — Victoria Abramchenko, Deputy Prime Minister of the Russian Federation.
COVID has clearly showed us all that we are only strong when united. Global threats require global solutions. Climate now poses one of the greatest challenges — Selwin Charles Hart, Special Adviser to the Secretary-General on Climate Action, United Nations.
The material was prepared by the Russian news agency TASS

redpajama_set_name: RedPajamaCommonCrawl


text:
Eswatini National Provident Fund
YOUR FUTURE, YOUR LIFE!
what plans have you made for retirement.
Save for your family
Ingungu
The Eswatini National Provident Fund was established in 1974 as a retirement savings scheme, the main purpose of which is to provide benefits for employed persons when they retire from regular employment in old age or in the event of becoming incapacitated.
All employers of labour in Eswatini are required by law to become contributing members of the Fund, and must pay a contribution for every eligible staff member. The employee's share (one half of the stipulated amount) is deducted from wages.
Disability Benefit
Benefits can be paid to a member who has the misfortune to become permanently unemployable through physical or mental disability; incapacity for work is assessed having regard to medical factors...read more.
Emigration Benefit
If a member satisfies the Chief Executive Officer that he is about to emigrate, or has emigrated from Eswatini with no intention of returning to the Kingdom, an emigration benefit is payable without regard to age. ...read more.
Survivor's Benefit
If a member dies before qualifying for a personal benefit, the amount in his benefit account as available to surviving members of his family as a survivor's benefit. Such a benefit is payable in accordance with the provisions of the ENPF Order No. 23 of 1974....read more.
Age Benefit
Benefit on age grounds is paid when the member is 50 years of age, but it can be claimed before then as a "retirement benefit" if the member is at least 45 years of age and has retired from regular employment or become self-employed....read more.
Imbube King's Marathon
The annual Imbube Marathon returns for its fourth instalment
Stakeholder Forum
The Stakeholder Annual General Meeting approaches...
ENPF Head Quarters
Lidlelantfongeni Building
Corner Martin & Ngwane Street, Manzini
ENPF NEWS
ENPF: COMPANIES CHOSE SCHOOLS THAT BENEFITTED FROM IMBUBE MARATHON
© 2020 by The Eswatini National Provident Fund.
Designed In-house


text:
Q: Encoding a list in Python based on order of occurance I have a list like this
['A', 'A', 'B', 'B', 'A+B', 'A+B', 'B', 'B', 'A']
I want to convert this list to like this
[1, 1, 2, 2, 3, 3, 4, 4, 5]
What I did is, I will start with value 1 and when there is a change in value from the list I will increment the value in hand by one.
This is the code that I am using now,
counts = []
count = 1

for i in range(1, len(p)):
    print(p[i-1], p[i])
    if p[i-1] == p[i]:
        counts.append(count)
    else:
        counts.append(count)
        count += 1

if p[-2] == p[-1]:
    counts.append(counts[-1])
else:
    counts.append(counts[-1] + 1)

How to do this in Python efficiently.? I can also make use of numpy if necessay

A: This is a simple way to do that with NumPy:
import numpy as np

data = np.array(['A', 'A', 'B', 'B', 'A+B', 'A+B', 'B', 'B', 'A'])
result = np.concatenate([[1], 1 + np.cumsum(data[:-1] != data[1:])])
print(result)
# [1 1 2 2 3 3 4 4 5]

Or with Pandas:
import pandas as pd

data = pd.Series(['A', 'A', 'B', 'B', 'A+B', 'A+B', 'B', 'B', 'A'])
result = data.ne(data.shift(1)).cumsum()
print(result)
# 0    1
# 1    1
# 2    2
# 3    2
# 4    3
# 5    3
# 6    4
# 7    4
# 8    5
# dtype: int32

redpajama_set_name: RedPajamaStackExchange


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text:
Neo-sex Chromosomes in the Monarch Butterfly, Danaus plexippus
Andrew J. Mongue, Petr Nguyen, Anna Voleníková and James R. Walters
G3: Genes, Genomes, Genetics October 1, 2017 vol. 7 no. 10 3281-3294; https://doi.org/10.1534/g3.117.300187
Andrew J. Mongue
Department of Ecology and Evolutionary Biology, University of Kansas, Lawrence, Kansas 66046
Petr Nguyen
Faculty of Science, University of South Bohemia in České Budéjovice, 370 05 České Budějovice, Czech RepublicInstitute of Entomology, Biology Centre of the Czech Academy of Sciences, Czech Academy of Sciences, 370 05 České Budějovice, Czech Republic
Anna Voleníková
James R. Walters
For correspondence: jrwalters@ku.edu
We report the discovery of a neo-sex chromosome in the monarch butterfly, Danaus plexippus, and several of its close relatives. Z-linked scaffolds in the D. plexippus genome assembly were identified via sex-specific differences in Illumina sequencing coverage. Additionally, a majority of the D. plexippus genome assembly was assigned to chromosomes based on counts of one-to-one orthologs relative to the butterfly Melitaea cinxia (with replication using two other lepidopteran species), in which genome scaffolds have been mapped to linkage groups. Sequencing coverage-based assessments of Z linkage combined with homology-based chromosomal assignments provided strong evidence for a Z-autosome fusion in the Danaus lineage, involving the autosome homologous to chromosome 21 in M. cinxia. Coverage analysis also identified three notable assembly errors resulting in chimeric Z-autosome scaffolds. Cytogenetic analysis further revealed a large W chromosome that is partially euchromatic, consistent with being a neo-W chromosome. The discovery of a neo-Z and the provisional assignment of chromosome linkage for >90% of D. plexippus genes lays the foundation for novel insights concerning sex chromosome evolution in this female-heterogametic model species for functional and evolutionary genomics.
chromosomal fusion
Major rearrangements of karyotype and chromosome structure often have substantial evolutionary impacts on both the organisms carrying such mutations and the genes linked to such genomic reorganization (Lynch and Walsh 2007; Soltis and Soltis 2012). Additionally, such large-scale chromosomal mutations often present novel opportunities to investigate molecular, evolutionary, and functional genetic processes, for instance the evolution of neo-sex chromosomes, which can arise from the fusion of an autosome with an existing and well-differentiated allosome. This effectively instantaneous transformation of a formerly autosomal set of genes into sex-linked loci is fertile ground for advancing our understanding of the distinct set of evolutionary forces acting on sex chromosomes relative to autosomes (Bachtrog et al. 2009; Pala et al. 2012a; Bachtrog 2013; Šíchová et al. 2013). Furthermore, when such an event is observed in a tractable genetic model system, there is opportunity to explore the functional and mechanistic changes associated with sex chromosome evolution. The congruence of neo-sex chromosomes existing in a model system is relatively rare, although there are some notable examples. For example, independent origins of neo-sex chromosomes are known in Drosophila fruit flies (Counterman et al. 2004; Flores et al. 2008; Bachtrog et al. 2009; Zhou et al. 2013; Brown and Bachtrog 2014; Nozawa et al. 2014) and stickleback fish, where neo-sex chromosomes appear to play an important role in reproductive isolation between incipient species (Kitano et al. 2009; Yoshida et al. 2014; White et al. 2015).
Looking beyond these established model systems, the rapid expansion of genomic technologies has allowed extensive analyses of gene content, sex-biased gene expression, dosage compensation, and sequence divergence for recently evolved sex chromosomes among a very diverse set of organisms including several insect lineages [teleopsid flies, a grasshopper, and Strepsiptera (Baker and Wilkinson 2010; Mahajan and Bachtrog 2015; Palacios-Gimenez et al. 2015)], vertebrates [mammals and birds (Zhou et al. 2008; Pala et al. 2012a; Murata et al. 2015)], and plants [Silene and Rumex genera (Hough et al. 2014; Charlesworth 2015; Papadopulos et al. 2015)]. A clear consensus emerges from this research that the lack of recombination associated with sex chromosomes catalyzes a cascade of evolutionary changes involving the degeneration of one allosome, the accumulation of genes with sex-biased expression, increased evolutionary rates, and often, but not always, the acquisition of dosage compensation. Yet many of the details in this process remain elusive and unresolved, including the rate of allosome divergence, the role of positive selection vs. drift, the importance of sex-specific selection, and the mechanisms underlying dosage compensation or the reasons for its absence. It is therefore important to continue identifying new opportunities for novel insight into the evolution of sex chromosomes.
Overwhelmingly, research on sex chromosomes occurs in male-heterogametic (XY) species (Vicoso and Charlesworth 2006; Ellegren 2011; Bachtrog 2013; Parsch and Ellegren 2013). This is particularly true for neo-sex chromosomes, where contemporary genomic analyses of neo-Z or neo-W chromosomes are currently lacking, with one notable exception for birds (Pala et al. 2012a). This imbalance is unfortunate, because WZ sex determination offers the novel framework of female-specific selection during the evolution of heterogamety and is a common form of sex determination in both vertebrates and invertebrates. Birds are the most prominent vertebrate taxon that is female heterogametic, but it appears that avian neo-sex chromosomes are quite rare and indeed absent from prominent model species, e.g., chicken, zebra finch (Nanda et al. 2008; Pala et al. 2012b). Fishes and squamates seem to be far more labile in sex chromosome constitution, with numerous independent transitions between male and female heterogamety and relatively frequent sex–autosome fusions (Pennell et al. 2015), thus there are potentially great opportunities in these taxa. However, no tractable WZ model system with neo-sex chromosomes has been identified for these lineages.
For many reasons, Lepidoptera (moths and butterflies) may be the most promising female-heterogametic taxon for studying neo-sex chromosomes. Synteny, i.e., the chromosomal placement of orthologous genes between species, is unusually well conserved in Lepidoptera (Pringle et al. 2007; The Heliconius Genome Consortium 2012; Ahola et al. 2014; Kanost et al. 2016); yet there are also numerous known examples of independently evolved neo-Z and neo-W chromosomes, several of which have been well characterized cytogenetically (Traut et al. 2007; Yoshido et al. 2011; Nguyen et al. 2013; Šíchová et al. 2013; Smith et al. 2016). Furthermore, comparative genomic resources in this insect order are substantial and growing quickly (Challis et al. 2016).
In this context, we report the discovery of a neo-Z chromosome in the monarch butterfly, Danaus plexippus, and closely related species. Monarch butterflies, renowned for their annual migration across North America, already have a strong precedent as a model system in ecology (Urquhart 1976; Oberhauser and Solensky 2004). Recently, monarchs have emerged as a model system for genome biology, with a well-assembled reference genome, extensive population resequencing data, and a precedent for genome engineering (Zhan et al. 2011, 2014; Merlin et al. 2013; Markert et al. 2016). The discovery of a neo-Z chromosome further enriches the value of this species as a research model in genome biology and lays the foundation for extensive future insights into the evolution and functional diversity of sex chromosomes.
Sequencing coverage analysis
Illumina shotgun genomic DNA (gDNA) sequencing data for three male and three female D. plexippus individuals were selected for analysis from samples sequenced by Zhan et al. (2014). Details of sample identities, including GenBank SRA accessions, are given in Supplemental Material, Table S1 in File S1. Male–female pairs were selected on the basis of approximately equal sequencing coverage. Samples were aligned to the D. plexippus version 3 genome assembly with Bowtie2 (v2.1.0), using the "very sensitive local" alignment option (Langmead and Salzberg 2012; Zhan and Reppert 2013). The resulting alignments were parsed with the genomecov and groupby utilities in the Bedtools software suite (v2.17.0) to obtain a per-base median coverage depth statistic for each scaffold (Quinlan and Hall 2010). Genomic sequencing data from other Danaus species, also generated by Zhan et al. (2014), were aligned to the same assembly using Stampy (v1.0.22) (default parameters, except for substitutionrate = 0.1) (Lunter and Goodson 2011).
Coverage analyses comparing males and females were limited to scaffolds of lengths equal to or greater than the N90 scaffold (160,499 bp) (Zhan and Reppert 2013). Also, incomplete cases were excluded (i.e., scaffolds with no reads from one or more samples). In total, 140 scaffolds were excluded, leaving 5257 scaffolds analyzed. For each sample, each scaffold's median coverage was divided by the mean across all scaffold median coverages, thereby normalizing for differences in overall sequencing depth between samples. Samples were grouped by sex and the per-scaffold mean of normalized coverage depth was compared between sexes, formulated as the log2 of the male:female (M:F) coverage ratio. Autosomal scaffolds are expected to exhibit equal coverage between sexes, yielding a log2 ratio of zero. Z-linked scaffolds should have a ratio of one, due to the twofold-greater representation in males. Manipulation, analysis, and visualization of coverage data were performed in R (R Developement Core Team 2015).
A few scaffolds yielded intermediate M:F coverage ratios falling between equality and a twofold difference. We further examined these scaffolds using Bedtools genomecov to calculate per-base coverage, in order to identify regions with conspicuous shifts in coverage that may indicate potential assembly errors producing Z-autosomal chimeric scaffolds. For each sample, coverage per base was divided by the mean of all scaffold median coverage values, thus normalizing for overall sequencing depth. The normalized coverage per base was averaged within sex and visualized along the length of the scaffold by using the median of a 5-kbp sliding window, shifted by 1-kbp steps.
Point estimates for Z-autosomal break points in chimeric scaffolds were generated using a sliding window analysis of M:F coverage ratios. Putative break points were obtained as the maximum of the absolute difference between adjacent nonoverlapping windows. A window of 150 kbp with 10-kbp steps was used for DPSCF300001 and the 5′ break point of DPSCF30028. A window of 10 kbp with 1-kbp steps was used for DPSCF30044, and in a second, localized analysis between 1.5 Mb and the 3′ terminus of DPSCF30028 to localize the second, 3′ break point.
Potential W-linked scaffolds
After the identification of the neo-Z chromosome (see Results), a post hoc search was conducted for W-linked scaffolds in the genome assembly. The D. plexippus genome assembly was primarily generated with data from a female individual (Zhan et al. 2011), thus it is possible that W-linked scaffolds are present in the genome assembly. Such scaffolds should have strongly female-biased patterns of sequencing coverage, but may also be quite short and even completely lack male coverage. We therefore reexamined the normalized coverage data as above for all scaffolds, ignoring previous criteria for length or coverage values. Scaffolds with more than twofold-greater average coverage in females were considered potentially W-linked.
Potential W scaffolds were aligned to Z-linked scaffolds via six-frame amino acid translations using the PROmer algorithm and visualized with mummerplot, both from the MUMmer software package (v3.1) (Kurtz et al. 2004). Predicted proteins from potentially W-linked scaffolds were searched against the all D. plexippus genomic scaffolds via tBLASTn and also against the GenBank nonredundant protein database via BLASTp.
Orthology-based chromosomal assignments for D. plexippus scaffolds
Putative chromosomal linkage was predicted for D. plexippus scaffolds relative to the genome assemblies of three reference species, Melitaea cinxia, Bombyx mori, and Heliconius melpomene (the Glanville fritillary, domestic silkmoth, and postman butterfly), based on counts of orthologous genes (The International Silkworm Genome Consortium 2008; The Heliconius Genome Consortium 2012; Ahola et al. 2014). Orthologous proteins were predicted between D. plexippus and each reference species using the Proteinortho pipeline (Lechner et al. 2011). Using only one-to-one orthologs, we tabulated per D. plexippus scaffold the number of genes mapped to each chromosome in the reference species. Each D. plexippus scaffold was assigned to the chromosome with the highest count of orthologs in the reference species. Scaffolds were excluded from analysis when maximum ortholog count was tied between two or more scaffolds, though this situation was rare and only involved scaffolds with low genes counts.
Point estimate of the Z-autosome fusion
The fusion point in monarch between ancestrally Z and autosomal segments was localized by aligning the homologous H. melpomene or M. cinxia chromosomes against monarch scaffold DPSCF300001 (Ahola et al. 2014; Davey et al. 2016). Alignments were based on six-frame amino acid translations using the PROmer algorithm and visualized with mummerplot, both from the MUMmer software package (v3.1) (Kurtz et al. 2004). We initially aligned the complete set of scaffolds from the Z (HmChr21, McChr1) or relevant autosome (HmChr2, McChr21), yielding a preliminary indication that the Z-autosome fusion point occurred at ∼4 Mbp on DPSCF300001. To refine and better visualize this phenomenon, pseudoassemblies were created for each chromosome using query scaffolds producing >500 bp of total aligned coverage on DPSCF300001. Selected query scaffolds were concatenated into a single FASTA entry, with ordering based on target alignment positions. For each species, the Z and autosomal pseudoassemblies were coaligned to DPSCF300001. The transition point between contiguous alignments of the two pseudoassemblies from distinct chromosomes was interpreted as the approximate location of the Z-autosome fusion in monarch.
Cytogenetic analysis
All D. plexippus tissues used for cytogenetic analysis were from captive-bred butterflies reared on an artificial diet provided by Monarch Watch (MonarchWatch.org). Spread chromosome preparations were made from gonads of third to fifth instar larvae of both sexes following Mediouni et al. (2004). To test for the presence of sex chromatin, preparations of polyploid somatic nuclei were made, according to Traut et al. (1986), from Malpighian tubules dissected from the same material.
gDNA was isolated separately from males and females by standard phenol-chloroform extraction. Briefly, larval tissues were homogenized in liquid nitrogen, transferred in lysis buffer (100 mM NaCl, 10 mM Tris-HCl, pH 8.0, 50 mM EDTA, 100 μg/ml Proteinase K, 0.5% Sarkosyl), and incubated at 37° overnight. The samples were then treated with RNase A (10 µg/ml) and purified by three phenol, one phenol-chloroform, and one chloroform extractions. Hybridization probes were prepared from the entire complement of extracted male or female gDNA, which was labeled by aminoallyl-dUTP-Cy3 or fluorescein-12-dUTP (both Jena Bioscience GmbH, Jena, Germany) using the Nick Translation Kit (Abbott Molecular Inc., Des Plaines, IL) with 7 hr incubation at 15° (Šíchová et al. 2015).
Genomic in situ hybridization (GISH) was performed as described by Fuková et al. (2005). Comparative genomic hybridization (CGH) was carried out according to the protocol in Šíchová et al. (2013) with several modifications, as follows. Prior to denaturation, RNase A-treated slides were incubated in 5× Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin) at 37° for 30 min. A 10-µl hybridization mixture consisting of labeled female and male probes (350 ng each), sonicated salmon sperm DNA (25 µg), 50% deionized formamide, and 10% dextran sulfate in 2× SSC was denatured and allowed to reanneal at 37° for 2 hr (cf. Kallioniemi et al. 1992) before it was hybridized to the denatured female preparation.
Results were documented in a Zeiss Axioplan 2 Microscope (Carl Zeiss, Jena, Germany) equipped with the appropriate fluorescence filter set. Images were captured with an Olympus CCD Monochrome Camera XM10 equipped with cellSens 1.9 digital imaging software. The images were pseudocolored and superimposed with Adobe Photoshop CS3.
Putative chromosomal assignments for D. plexippus scaffolds and genes are provided in Table S3 in File S2. Estimated coordinates for break points reported for chimeric Z-autosomal assemblies are provided in Table S4 in File S2. Other intermediate results files and code used in the described analyses are at https://github.com/WaltersLab/Danaus_NeoZ-Public.
Identifying Z-linked scaffolds in D. plexippus
We identified Z-linked scaffolds in the D. plexippus genome assembly (Zhan et al. 2011; Zhan and Reppert 2013) by comparing sequencing coverage from male and female samples. Males should have twice the Z-chromosome DNA content than females, while autosomes should have equal DNA content between sexes. Thus a corresponding twofold difference in sequencing coverage is expected between sexes for the Z chromosome, but not autosomes, and this can be used to identify Z-linked scaffolds (Martin et al. 2013; Vicoso et al. 2013; Mahajan and Bachtrog 2015). A histogram of M:F ratios of median coverage clearly identifies two groups of scaffolds (Figure 1). One large cluster is centered around equal coverage between sexes (log2 M:F = 0) and a second, smaller cluster is centered around twofold-greater coverage in males (log2 M:F =1). We can thus clearly distinguish the Z-linked scaffolds as those with log2(M:F) >0.5, with the remainder of the scaffolds presumed to be autosomal.
Distribution of median normalized M:F genomic sequencing coverage ratios for D. plexippus version 3 assembly scaffolds. Only scaffolds of length equal to or greater than the N90 scaffold are shown. The dotted line at 0.5 represents the value used to partition scaffolds as autosomal (gray) or Z linked (red).
One scaffold, DPSCF300028, appeared to have an intermediate coverage ratio, falling at log2 M:F ≈ 0.7. One likely explanation for such an intermediate value is that the scaffold is a chimera of Z-linked and autosomal sequence arising from an error in genome assembly (Martin et al. 2013). In this scenario, only a portion of the scaffold is Z-linked and gives a twofold difference in coverage between sexes; the remaining autosomal fraction of the scaffold yields equal coverage. The resulting estimate of average coverage for the entire scaffold then falls at a value between expectations for Z or autosomal scaffolds. This appears to be true for DPSCF300028, as revealed by examining base pair-level sequencing coverage across the scaffold (Figure 2A). While average male coverage is consistent across the entire length of the scaffold, female coverage exhibits a clear transition between coverage equal to males (the autosomal portion) and coverage one half that of males (the Z-linked portion). Indeed, there are two such transitions in scaffold DPSCF300028, which we estimate to occur at 0.76 and 1.805 Mbp, creating a Z segment flanked by autosomal segments.
Normalized male and female coverage along the length of chimeric scaffolds, for (A) DPSCF300028, (B) DPSCF300044, and (C) DPSCF300001. Coverages are plotted as sliding windows (width = 5 kbp, step = 1 kbp) of median base pair values. The associated M:F ratio of coverage for each window is plotted as a red line below the pair of sex-specific plots. * indicates the estimated break point between Z-linked and autosomal segments of each scaffold, as determined by the maximum difference in adjacent, nonoverlapping windows of M:F ratio (see Materials and Methods for details).
However, an alternative explanation for this pattern could be that (portions of) the Z and W retain substantial homology and that sequencing reads from the W are mapping to closely related regions of the Z. This is particularly likely in cases where there is a recently acquired neo-sex chromosome and limited time for divergent mutations to accumulate between homologous regions of the neo-Z and neo-W. Assessing patterns of homology relative to chromosomal linkage in other species is one useful way to further assess whether these scaffolds with intermediate levels of coverage reflect chimeric assemblies or sex-linked regions of reduced divergence between (neo-) Z and W chromosomes.
Ortholog counts link scaffolds to chromosomes
As mentioned above, Lepidoptera show a very high level of synteny conserved across substantial evolutionary distances (Pringle et al. 2007; The Heliconius Genome Consortium 2012; Ahola et al. 2014; Kanost et al. 2016). Thus, it is possible to use counts of orthologous genes to assign D. plexippus scaffolds to linkage groups (i.e., chromosomes) delineated in other moth or butterfly species. We generated predicted orthologs between D. plexippus and three other reference species where genetic linkage mapping has been used to assign genomic scaffolds to chromosomes: M. cinxia (n = 31), H. melpomene (n = 21), and B. mori (n = 28) (The International Silkworm Genome Consortium 2008; The Heliconius Genome Consortium 2012; Ahola et al. 2014). M. cinxia and H. melpomene are both butterflies equally diverged from D. plexippus and all three are part of the same family, Nymphalidae; the silkmoth, B. mori, is distinctly more diverged, located outside of the suborder containing all butterflies (Wahlberg et al. 2009; Kawahara and Breinholt 2014).
To assign D. plexippus scaffolds to chromosomes, we tabulated per scaffold the counts of one-to-one reference species orthologs per reference species chromosome. D. plexippus scaffolds were then assigned to the reference chromosome with the maximum count of orthologs. For a few scaffolds, a tie occurred in maximum ortholog count per reference chromosome, in which case the scaffold was removed from further analysis; at most this occurred for only 14 scaffolds per reference species and usually involved small scaffolds harboring fewer than five orthologs. Typically, this method yielded a single obvious reference chromosomal assignment for each D. plexippus scaffold.
This method of ortholog-count, chromosomal "lift-over" resulted in putative chromosomal assignments for >90% of D. plexippus genes relative to each reference species (Table 1 and Table S2 in File S1). Also, more than 4500 orthologous genes were colocalized to a chromosome between D. plexippus and each reference species. Having several thousand orthologs mapped to a chromosome in D. plexippus and a reference species presents the opportunity to examine the extent of chromosomal rearrangements and gene movement between the two species. Here we primarily report the comparison with M. cinxia because this species is believed to retain the ancestral lepidopteran karyotype of 31 chromosomes (Ahola et al. 2014). Furthermore, this count of chromosomes is closest to that reported for several Danaus butterflies, including monarch (n = 30, see Figure 3), making it the most appropriate comparison available (Brown et al. 2004). H. melpomene and B. mori are known to have less similar karyotypes involving several chromosomal fusions relative to M. cinxia; nonetheless, details of comparisons to these two species are reported in the supplementary content (Figure S1, Figure S2, and Table S2 in File S1) and provide comparable support for the primary findings reported here.
Table 1 Summary of assigning D. plexippus genes and scaffolds to chromosomes via orthology lift-over relative to M. cinxia
Chromosomal colinkage between D. plexippus and M. cinxia for predicted orthologous proteins.
The cross-tabulation of chromosomal linkage for >4500 orthologs between M. cinxia and D. plexippus is summarized in Figure 3. The overwhelming majority of orthologs fall on the diagonal, suggesting substantial conservation of chromosomal linkage and relatively little gene shuffling, which appears consistent with other analyses of gene movement in Lepidoptera (The Heliconius Genome Consortium 2012; Ahola et al. 2014; Kanost et al. 2016). However, because this lift-over approach is based on an assumption of conserved synteny and does not include an independent assessment of chromosomal linkage in monarch, it does not exclude the possibility that large-scale changes in synteny remain to be detected. Nonetheless, two notable exceptions to the general pattern of conservation emerged, both involving the Z chromosome (Chr1). In one case (McChr9, DpChr1) we could anticipate this because of the previously identified scaffold, DPSCF300028, with intermediate-average coverage and a sharp transition in base-level depths of sequencing. This scaffold harbors 34 orthologs assigned to McChr1 and 23 orthologs assigned to McChr9. This irregular pattern of M:F coverage ratios seems most readily explained by a chimeric Z-autosome assembly, though we cannot exclude the possibility of a recent insertion from McChr9 which retains regions of substantial similarity between sex chromosomes (Figure 2A).
The second case (McChr1, DpChr21) appeared to arise entirely from a single scaffold, DPSCF300001, the largest scaffold in the D. plexippus v3 assembly. This scaffold carried 107 orthologs assigned to McChr21, 28 orthologs assigned to McChr1, 13 orthologs assigned to McChr23, and a few other orthologs assigned to other autosomes. Notably, despite the large number of apparently autosomal orthologs, the average M:F coverage ratio for DPSCF300001 was consistent with it being Z-linked [log2(M:F coverage) = 0.92]. Nonetheless, we plotted coverage across the chromosome and detected a ∼1-Mbp portion at the 3′ end of the scaffold with coverage patterns consistent with being an autosome (Figure 2C). The M. cinxia orthologs in this apparently autosomal portion, with an estimated break point at 5.82 Mbp, were linked exclusively to McChr23. There was not an obvious shift in sequencing coverage between sexes to indicate a misassembled Z-autosome chimera involving McChr21. Rather, it appeared that nearly the entirety of scaffold DPSCF300001 had twice the coverage in males than in females, consistent with Z linkage for regions apparently homologous both to McChr1(Z) and McChr21.
A neo-Z chromosome in D. plexippus
The observation that a substantial portion of scaffold DPSCF300001 was Z-linked and homologous to McChr21, while another large section of the same scaffold was homologous to McChr1, i.e., McChrZ, led us to hypothesize that a Z-autosome fusion could readily explain the karyotypic differences between D. plexippus (n = 30) and M. cinxia (n = 31). To further investigate this hypothesis of a major evolutionary transition in sex chromosome composition in the Danaus lineage, we examined the chromosomal assignments for all monarch scaffolds identified as Z linked via sequencing coverage ratios (Z-cov scaffolds). Specifically, we identified the unique set of reference chromosomes to which Z-cov scaffolds were assigned, and then examined the M:F coverage ratio for all scaffolds assigned to those reference chromosomes. In the case of M. cinxia as the reference, all Z-cov scaffolds were assigned either to McChr1 or McChr21 (Figure 4; comparable results were obtained for H. melpomene and B. mori, Figure S2 in File S1). This result provides further evidence that the Z in D. plexippus is a neo-sex chromosome reflecting the fusion of the ancestral Z chromosome with an autosome homologous to McChr21.
Ratios of M:F, median-normalized, genomic sequencing coverage plotted against scaffold length. Scaffolds homologous via lift-over procedure to M. cinxia chromosomes 1/Z (blue) and 21 (green) are plotted in distinct colors. Dotted lines indicate expected values for Z-linked (red) and autosomal (black) scaffolds.
This analysis intersecting Z-cov scaffolds with homology to M. cinxia revealed two scaffolds that did not fit with the expected pattern of sequencing coverage (Figure 5). First, scaffold DPSCAF300044 was assigned to McChr1(Z) but had log2 M:F ≈ 0.25, much more like other autosomes than other Z-linked chromosomes. This scaffold had seven Z-linked orthologs and four autosomal ones, suggesting another chimeric scaffold. Indeed, examining coverage across the scaffold revealed a clear transition in coverage as previously observed for DPSCF300001 and DPSCF300028 (Figure 2B). Thus the low M:F coverage ratio for this scaffold could be the artifact of an assembly error. Alternatively, the M:F coverage ratio could reflect reduced divergence between neo-Z and neo-W, with neo-W-linked reads mapping to the Z scaffold. Examining base-level coverage, we were again able to identify a sharp transition in coverage levels that partitioned the scaffold into two sections. One section was consistent with autosomal (i.e., equal) M:F coverage and the other with Z-linked (i.e., twofold) M:F coverage, with a break point estimated at 0.29 Mbp from the 5′ end. The autosomal section contained approximately equal counts of orthologs assigned to two distinct autosomes in M. cinxia, as well as in the other reference species analyzed, so linkage to a specific autosome could not be predicted. Nonetheless, homology to autosomes other than McChr21 for the portion of DPSCAF300044 with equal M:F coverage supports the notion of a chimeric assembly over retained allosome homology. Table S3 in File S2 summarizes break points and predicted scaffold assignments for the three chimeric Z-autosome scaffolds identified here.
Ratios of M:F, median-normalized, genomic sequencing coverage plotted by scaffold length for four species of Danaus butterflies. The dotted line at log2(M:F) = 0.5 represents the threshold used to discern autosomal (<0.5) from Z-linked (>0.5) scaffolds.
DPSCF300403 was the other scaffold tentatively identified as homologous to McChr21, but without the characteristic twofold difference in M:F coverage. Rather, the M:F coverage ratio along the scaffold was approximately equal, consistent with it being entirely autosomal (Figure S3 in File S1). However, it may also be that retained sex-linked homology could produce this coverage pattern. In this case, the scaffold carried only a single one-to-one orthologous gene (and only five protein-coding genes in total), so the assignment to McChr21 is rather tenuous. This scaffold also had a single one-to-one ortholog found in B. mori, and none identified in H. melpomene. Given the quite limited evidence of chromosomal homology for this scaffold, we thus consider DPSCF300403 essentially uninformative concerning the presence of neo-sex chromosomes in D. plexippus.
The neo-Z chromosome exists in the monarch's close relatives
The monarch population genomic data set from Zhan et al. (2014) also contained male and female resequencing samples from three closely related congeners: D. gilippus, D. erippus, and D. eresimus. This allowed us to assess whether this neo-Z exists in these related species similarly to D. plexippus. Analyzing male vs. female resequencing in these species does indeed show the same scaffolds homologous to both McChr1 and McChr21 as having coverage differences consistent with a neo-Z (Figure 5). Thus it appears that the origin of this neo-Z predates the diversification of the genus Danaus.
Annotating chromosomal linkage
The combination of sequencing coverage analysis and comparative lift-over allowed us to provisionally assign most genes to chromosomes in D. plexippus. Genes falling on Z-cov scaffolds, or within the portion assessed as Z linked for noted chimeric scaffolds, have been assigned to the Z chromosome. We further partitioned these Z-linked genes into being on the ancestral (anc-Z) or neo- (neo-Z) portion of the Z, based on scaffold homology to reference chromosomes. In the case of DPSCF300001, we localized the fusion point between anc-Z and neo-Z by aligning M. cinxia and H. melpomene scaffolds from the Z (HmChr21, McChr1) or relevant autosome (HmChr2, McChr21). Alignments with both species were consistent in placing the fusion point at ∼3.88 Mbp from the 5′ end of the scaffold (Figure S4). Otherwise, genes and scaffolds were assigned to chromosomes based directly on the results of the lift-over relative to M. cinxia. Table 2 gives a tabulated summary of results, while results for every protein-coding gene are provided in Table S4 in File S2.
Table 2 Summary of provisional chromosomal linkage for D. plexippus protein-coding genes, with chromosomal identity reflecting homology to M. cinxia
Preparations of highly polyploid nuclei of Malpighian tubules from D. plexippus larvae were examined for the presence of a so-called "sex chromatin," i.e., a female-specific heterochromatin body consisting of multiple copies of the W chromosome (Traut and Marec 1996). Large multi-lobed nuclei were observed on both male and female preparations (Figure 6), which suggests a high degree of polyploidy in the examined cells, as expected in Malpighian tubules (cf. Buntrock et al. 2012). All female nuclei contained a single, highly stained heterochromatin body (Figure 6A). In contrast, no such heterochromatin was detected in male somatic nuclei (Figure 6B). This discrepancy confirms the female specificity of the heterochromatin observed, and indicates a W chromosome is a component of the D. plexippus genome.
Multi-lobed, highly polyploid nuclei of the Malpighian tubules from D. plexippus larvae stained by orcein. (A) A female nucleus with a single deeply stained sex chromatin body (arrow). (B) A male polyploid nucleus with no heterochromatin. Bar, 20 µm.
Spread mitotic complements of males contained 2n = 60 chromosomes. The chromosomes were generally small and uniform, as is typical of lepidopteran karyotypes (Marec et al. 2009), except for two distinctly larger elements (Figure 7A). Female mitotic metaphase consisted of 2n = 60 elements as well. In females, however, the two largest chromosomes differ in intensity of their DAPI staining. The deeply stained element found exclusively in females presumably represents the W sex chromosome consisting of AT-rich heterochromatin (Figure 7B; Kapuscinski 1979).
Cytogenetic analysis of sex chromosomes in D. plexippus. Chromosomes were counterstained by DAPI (blue). (A) Male mitotic metaphase complement consisting of 2n = 60 elements. Note the large chromosome pair (arrowheads). (B) Female mitotic metaphase nucleus comprising 2n = 60 chromosomes. The largest elements differ in intensity of their staining (arrowhead and arrow, the latter marking highly stained chromosome). (C–G) Female complements examined by GISH. Female-derived probe was labeled by Cy3 (red). (C) The same mitotic complement as in (B). The probe identified the DAPI-positive chromosome as a W chromosome and thus indicated the other large chromosome to be the Z chromosome. (D) Female pachytene complement consisting of 30 bivalents. The WZ bivalent was clearly recognized by female-derived probe, which highlighted whole W chromosome except its terminal segments. The probe also marked three terminal regions and one interstitial region of several autosomes (*). (E) The WZ bivalent stained by DAPI. Note that only about two thirds of the W chromosome thread are deeply stained and apparently heterochromatic. The more lightly stained euchromatic segment is marked by an arrow. (F) Hybridization signal of female-derived probe highlighting the W chromosome thread. Note that the signal is weaker in the euchromatic region (arrow). (G) Composite image of DAPI and the probe. (H–L) A pachytene WZ bivalent probed by CGH. Male-derived probe was labeled by Cy3 (red), female-derived probe by fluorescein (green). (H) The WZ bivalent stained by DAPI. The weakly stained Z is wrapped around the strongly stained W, which is folded in half. (I) Hybridization signal of female-derived probe. Note that the probe labeled only a small region of the euchromatic segment (arrow). (J) Hybridization signal of male-derived probe strongly highlights two W segments (arrows). (K) Both the female- and male-derived signals merged. (L) Composite image of DAPI and both probes. Bars, 5 µm in (A–G) and 2.5 µm in (H); (E–G) and (H–L) have the same scale.
To identify the W chromosome, we used GISH in which a probe generated by fluorescently labeling female gDNA is hybridized to female chromosome preparations in presence of excess of unlabeled male competitor gDNA. The male competitor gDNA binds probe sequences common to both sexes (e.g., autosomal and Z linked) and does not interact with the prepared sample. The remaining female gDNA probe hybridizes to regions containing female-specific and -enriched sequences, clearly differentiating the W chromosome in both mitotic and pachytene nuclei (Figure 7, C and D). GISH confirmed that the W chromosome is one of the two exceptionally large chromosomes in the D. plexippus karyotype (Figure 7C). In pachytene oocytes, the WZ bivalent was easily discernible by the heterochromatic W chromosome thread. However, about one third of the chromosome was not strongly highlighted by DAPI and the intensity of its staining was comparable to autosomes, indicating a substantially euchromatic portion of the W that is consistent with being a neo-W (Figure 7E). Accordingly, the female-derived probe did not highlight the W chromosome homogeneously; the signal was weaker on both its ends and the euchromatic segment (Figure 7, E and F). Female-derived probes in GISH also strongly labeled one interstitial and a few terminal regions of some autosomes, which most likely contain clusters of repetitive sequences (Figure 7D).
CGH is used to assess the broad molecular composition of the W chromosome (Sahara et al. 2003). It is based on the same principle as GISH. In this method, however, the same amount of male and female gDNA labeled by Cy3 and fluorescein, respectively, is hybridized to preparations. In D. plexippus, the hybridization signal of female-derived probe labeled by fluorescein was largely consistent with the results obtained by GISH. The signal highlighted nearly the entire W chromosome thread, with the exception of its termini and euchromatic segment, in which the probe detected only a small interstitial block (Figure 7, I, K, and L). The male-derived probe labeled by Cy3 provided a relatively weak hybridization signal, which was scattered along the W chromosome. This male probe highlighted only two regions: the W chromosome end opposite to the euchromatic segment and the region highlighted within the euchromatin by the female-derived probe (Figure 7, J–L). Both probes also detected the same autosomal regions as GISH (data not shown).
Potential W scaffolds
In searching for W-linked scaffolds, we were primarily concerned with identifying sequences that would be informative regarding the presence of a neo-W chromosome. We identified 12 scaffolds with average coverage patterns suggestive of W-linkage. These scaffolds ranged in length from ∼4 to 180 kbp and collectively harbored a total of 17 predicted protein-coding loci. However, none of the scaffolds showed obvious homology to any Z-linked scaffolds. Only six of the 17 loci produced significant (e-value <10−5) tBLASTn hits to any Z-linked scaffold. In each case, the protein also had at least one, and typically many, stronger hits to autosomal loci. Thus, of primary significance concerning the presence of a neo-W chromosome, none of these potentially W-linked scaffolds showed evidence of homology to Z-linked scaffolds. A comprehensive description of results is provided in File S3.
Using a combination of genomic resequencing, comparative genomics, and cytogenetic analysis, we have documented the presence of a neo-Z chromosome in Danaus butterflies, along with what is likely an accompanying neo-W chromosome. This discovery of neo-sex chromosomes in Danaus butterflies and our discrimination of genes falling on the ancestral vs. recently autosomal portions of the Z are fundamental observations that provide the foundation for a host of future inferences. These results create novel opportunities to address rates of molecular evolution, the evolution of dosage compensation, the pattern of allosome divergence, and many other important questions in sex chromosome biology, all in a female-heterogametic species that is also an emerging genomic model system.
In analyzing patterns of chromosomal fusion in H. melpomene and B. mori relative to M. cinxia, Ahola et al. (2014) report a significant tendency for a limited set of ancestral chromosomes, particularly the smallest ones, to be involved in chromosomal fusion events. Neither the ancestral Z nor McChr21 are among these small, repeatedly fused chromosomes; thus the chromosomal fusion reported here does not fit neatly with this pattern. Nonetheless, HmChr2 (homologous to McChr21) is the second-smallest chromosome that remains unfused between these lineages (Davey et al. 2016). Consequently, it is also difficult to argue strongly that this Z-autosome fusion in Danaus is a striking contrast to the trend of chromosomal fusions involving small chromosomes.
Motivated by the bioinformatic discovery of a neo-Z chromosome, we performed cytogenetic analysis of the D. plexippus karyotype to provide further insight into evolution and molecular composition of the monarch sex chromosomes. Previously, an observation of n = 30 chromosomes was reported only for males (Nageswara-Rao and Murty 1975). [See Price (2017) for potential controversy over D. plexippus chromosome number, but note that Brown et al. (2004) states that "the number for D. plexippus comes from a population kept at the University of Madras, India." This note was overlooked by all and offers plausible explanation for D. plexippus in India.] Our current analysis confirms the same chromosome number not only in males but also in females (Figure 7, A and B). Furthermore, detailed analysis of mitotic complements revealed a large chromosome pair (Figure 7, A and B) and GISH clearly identified one chromosome of the pair as the W chromosome (Figure 7C). A similar, extraordinarily large chromosome pair was recently shown to correspond to neo-sex chromosomes in leaf-roller moths of the family Tortricidae (Nguyen et al. 2013; Šíchová et al. 2013).
GISH represents a simplified version of CGH, which has been successfully used for evaluating the gross molecular composition of lepidopteran W chromosomes (e.g., Mediouni et al. 2004; Fuková et al. 2005). Previous studies in several moth species contrasted fluorescence intensities of male- vs. female-derived probes and identified two common types of repeats on lepidopteran W chromosomes: (i) repetitive sequences common to both males and females, i.e., present in autosomes and Z chromosome; and (ii) repetitive sequences exclusively or predominantly present in females (Sahara et al. 2003). In these previously studied species, the W primarily contains the first type, i.e., ubiquitous repeats (e.g., Fuková et al. 2005; Šíchová et al. 2013). In contrast, the monarch W appears distinct from the W chromosomes of these other species because the majority of the monarch W chromosome overwhelmingly contains repeats of the second type, i.e., female-limited repeats. The monarch W was labeled primarily by the female-derived probe (Figure 7, I–K), indicating that it is primarily comprised of repetitive sequences either specific to or greatly enriched on the W chromosome. Only two small segments of the W showed notably high densities of ubiquitous repeats commonly enriched on the entire W in other lepidopteran species.
This discrepancy between monarch and other species could be related to the relatively small size of the D. plexippus genome. The monarch butterfly represents the smallest lepidopteran genome yet sequenced, with haploid nuclear content of male being 284 Mbp (see Doležel et al. 2003 for the conversion of pg of DNA to Mbp; Gregory and Hebert 2003) and female being 273 Mbp (Zhan et al. 2011). This small genome is presumably depleted of repetitive sequences found more ubiquitously among the autosomes and Z in other Lepidoptera. Indeed, repeat content constitutes only 13.1% of the D. plexippus genome assembly (Zhan et al. 2011). The results of CGH thus could reflect the fact that the W chromosome represents the last refuge for many such repetitive sequences in the monarch genome after otherwise being purged from the genome.
Cytogenetic analyses further indicate that the monarch W chromosome exhibits notable compositional heterogeneity. Both GISH and CGH revealed terminal gaps in female-derived signal (Figure 7, G and L). One plausible explanation for this lack of female-specific signals on the monarch W chromosome ends is that ectopic recombination homogenizes these subtelomeric chromosome regions. A similar GISH result was obtained in the codling moth, Cydia pomonella (Tortricidae), where the female-derived probe labeled the entire W chromosome except for both subtelomeric segments (Fuková et al. 2005). A related observation was made by Van't Hof et al. (2013), who proposed that a copy of the Z-linked laminin A gene was transferred to and maintained on a W chromosome of the peppered moth, Biston betularia (Geometridae), by gene conversion resulting from ectopic recombination between repeats localized in terminal chromosome regions. Thus it seems that this pattern of reduced differentiation in subtelomeric regions of the W chromosome is a relatively common phenomenon in Lepidoptera.
Another region distinctly identified by CGH corresponds to an interstitial block localized within a euchromatic chromosome segment. The block was illuminated by both female- and male-derived probes (Figure 7,J–L), which suggests presence of repetitive sequences common to autosomes and the Z chromosome (Sahara et al. 2003). This block, together with the adjacent terminal region, forms a chromosome segment with distinct molecular composition comprising about one third of the W chromosome. The monarch W thus shows a bipartite organization, with only two thirds of the chromosome being highly heterochromatic, while the remaining third appears euchromatic.
The identification of the neo-Z and putative neo-W in Danaus butterflies raises several questions about how, and when, these sex chromosomes arose. Concerning the sequence of events giving rise to the current karyotype, we can identify three possible scenarios resulting in equal chromosome numbers in males and females of D. plexippus. The first two scenarios begin with the fusion of the Z chromosome to the autosome corresponding to McChr21, giving rise to a multiple sex chromosome system of W1W2Z, and then followed either by (i) the loss of one W chromosome, or (ii) by the fusion of the W chromosomes. Alternatively, (iii) fusion of the ancestral W chromosome with the autosome corresponding to McChr21 resulted in a sex chromosome constitution of WZ1Z2 and was followed by fusion of the Z chromosomes. Given the large size of the monarch W chromosome relative to chromosomes other than the Z, which is of comparably large size, the first scenario would further demand considerable expansion of the W chromosome sequence. The observed combination of size and bipartite organization of the D. plexippus W chromosome seems more parsimonious with scenarios (ii) or (iii), i.e., a neo-W chromosome resulting from a W-autosome fusion occurring in addition to the neo-Z formation. However, caution is advised in this interpretation because W chromosome size can be misleading (Schartl et al. 2016) and cytogenetic examination of species bearing neo-Z chromosomes revealed considerable differences in the structure of their W chromosomes (Šíchová et al. 2013).
In the hope of further supporting this neo-W hypothesis, we investigated the possibility that W-linked scaffolds were present in the D. plexippus genome assembly [which was generated from a female sample (Zhan et al. 2011)]. Crucially, identifying W-linked sequence with obvious homology to the neo-Z would strongly indicate the presence of a neo-W chromosome. Unfortunately, we found no evidence for any such scaffolds; none of the potentially W-linked scaffolds identified via coverage patterns exhibited any notable similarity to Z-linked scaffolds. It is also possible that sufficient similarity exists between the neo-Z and a neo-W that substantial portions of the two would be collapsed into a single scaffold during genome assembly. For these regions/scaffolds, the M:F sequencing coverage would be strongly shifted toward equality. We did identify a few scaffolds with some homology to the Z or the fused autosome (e.g., McChr21) that presented intermediate-average coverage levels, with particular regions of nearly equal coverage between sexes. However, in almost every case, the region with balanced coverage also exhibited homology to chromosomes other than the one involved in the Z-fusion event. This is seemingly better explained as chimeric scaffolds resulting from assembly errors than by extensive allosome homology. The only potential exception to this was the ambiguous case of DPSCF300403, which could not be robustly assigned to a homologous chromosome. Ultimately, we could not identify any portion of the D. plexippus genome assembly that strongly supported the presence of a neo-W chromosome.
Nonetheless, these negative results are far from sufficient to exclude the presence of a neo-W in Danaus. If the neo-W is substantially degenerate and repeat rich, as seems to be the case, it would have been quite difficult to assemble, and thus very poorly represented, given the methods used for the current D. plexippus genome assembly. Nonetheless, determining whether and what protein-coding genes may remain on the W is of particular interest in this system. A promising route to further evaluate the presence of a neo-W would be via the application of novel long-read sequencing technologies and targeted laser capture of the W chromosome (Fuková et al. 2007; Chang and Larracuente 2017; Tomaszkiewicz et al. 2017).
Concerning the timing of these fusion events, our results suggest that the neo-sex chromosome system was present in a common ancestor of the four species examined, namely D. plexippus, D. gilippus, D. erippus, and D. eresimus. Given the phylogenetic relationships between Danaus species (Brower et al. 2010) and their chromosome numbers (Brown et al. 2004), the sex chromosome–autosome fusion appears to be a shared trait for the genus Danaus and therefore occurred at least 5 MYA (Lushai et al. 2003). However, we cannot exclude the possibility that the neo-sex chromosomes evolved even earlier in the evolution of the subtribe Danaini, as the karyotype n = 30 is often found elsewhere in the subtribe, shared by several species of the genera Danaus, Euploea, Idea, and Lycorea (Maeki and Ae 1968; Brown et al. 2004; Ahola et al. 2014). Nonetheless, assuming a minimum age of 5 MYA is helpful for contextualizing the Danaus neo-sex chromosomes in light of molecular analyses from other derived sex chromosome systems that are better analyzed.
Arguably the best comparison to neo-sex chromosomes in Lepidoptera is found among Drosophila fruit flies, which provide multiple cases of thoroughly studied neo-sex chromosomes of various ages. Importantly, in both Drosophila and Lepidoptera, meiosis in the heterogametic sex is achiasmatic, i.e., it occurs without recombination. Recombination between neo-sex chromosomes is thus restricted immediately and completely upon sex chromosome–autosome fusion and there is no opportunity for the formation of evolutionary strata or pseudoautosomal regions. For comparison, an age of 5 MY would place the Danaus neo-W roughly midway between the ages of the neo-Y chromosomes found in Drosophila pseudoobscura and D. miranda. We might therefore expect at least somewhat less degeneration on the Danaus neo-W than the D. pseudoobscura neo-Y, which formed in the range of 10–18 MYA and is now entirely heterochromatic with almost no detectable homology to the neo-X and only a handful of functional genes remaining (Carvalho and Clark 2005; Bachtrog 2013). In contrast, after (at least) 5 MY, degeneration of the Danaus neo-W chromosome has likely progressed far beyond the stage exemplified by the neo-Y of D. miranda, which formed ∼1 MYA. The D. miranda neo-Y chromosome is partially heterochromatic and has massively accumulated repetitive sequences. While the majority of neo-X-linked genes still have homologs on the neo-Y chromosome, nearly half of them are already pseudogenes (Zhou and Bachtrog 2012; Bachtrog 2013). Notably, Zhou and Bachtrog (2012) reported M:F ratios of read mapping coverage along the neo-X chromosome to be ∼3:4. In D. plexippus, sequencing data yield a very consistent 2:1 M:F coverage ratio on scaffold regions corresponding to McChr21. If a neo-W retained substantially close homology to the neo-Z, we would expect many sequencing reads emanating from the neo-W to align to the neo-Z, and shift this coverage ratio toward one. This evidently does not occur, suggesting substantial divergence between the neo-Z and any homologous neo-W sequence that is retained. The apparent absence of any neo-W scaffolds in the current genome assembly is consistent with substantial degeneration of a neo-W, assuming one exists.
Cytogenetic analyses confirm that the D. plexippus W chromosome is well differentiated relative to the Z (Figure 7, D–G). However, the presence of euchromatin on the W chromosome is intriguing and it is tempting to interpret its presence as indirect evidence of transcriptional activity of W-linked McChr21 orthologs. In general, heterochromatinization of a neo-W/Y chromosome involves two processes: (i) spreading of repetitive sequences which could serve as nucleation sites for initiating heterochromatin formation (Stanojcic et al. 2011); and (ii) loss of expression of W-linked genes, since expression generally attenuates the spread of heterochromatin (cf. Wheeler et al. 2009). Thus, presence of euchromatin could be due to low accumulation of certain types of repeats rather than ongoing gene expression. Analyses of contiguous W chromosome sequences are necessary to determine whether and how the protein-coding sequences are preserved on the W chromosome of D. plexippus (cf. Carvalho et al. 2015).
Finally, it should be noted that a relatively modern W-autosome fusion was recently reported to be segregating in the African queen butterfly, D. chrysippus, where it controls color pattern and male killing and is driving population divergence across a hybrid zone (Smith et al. 2016). Thus, the W-autosome fusion in D. chrysippus may represent a compound neo-W involving two distinct former autosomes. This pattern of relatively frequent karyotypic changes within the genus further recommends Danaus butterflies as an excellent model system for studying sex chromosome evolution.
We have used a combination of genome sequencing coverage, comparative genomic analysis, and cytogenetics to demonstrate that Danaus butterflies harbor a neo-Z chromosome resulting from the fusion of the ancestral Z chromosome and an autosome homologous to Chr21 in M. cinxia. Also, at least in the case of monarch butterflies, it appears plausible that a separate fusion event involving the W and this same autosome has resulted in a large neo-W chromosome with a prominent euchromatic region. Our analysis also identified and resolved several Z-autosome chimeric scaffolds in the most recent assembly of the D. plexippus genome. This discovery and provisional assignment of chromosomal linkage for >90% of D. plexippus genes paves the way for myriad and diverse investigations into sex chromosome evolution, which are likely to be of distinct importance given the increasing prominence of Danaus butterflies as a female-heterogametic model species for functional and evolutionary genomics.
Thanks to Chip Taylor, Ann Ryan, and the rest of MonarchWatch.org for donation of organisms used in this study. Jim Mallet and John Davey provided helpful comments on this work. This research was supported by NSF-DEB 1457758 to J.R.W. Cytogenetic analysis was supported by the Czech Science Foundation grants 14-35819P and 14-22765S. P.N. and A.V. are thankful for further support to the Fulbright program and grant 159/2016/P of the Grant Agency of the University of South Bohemia. The computing for this project was performed on the Community Cluster at the Center for Research Computing at the University of Kansas.
Author contributions: A.J.M., P.N., A.V., and J.R.W. all performed analyses and contributed to the manuscript text. A.J.M. additionally assembled analyses and manuscript components and edited the complete version. All authors read and approved the final manuscript. The authors declare no competing interests.
Supplemental material is available online at www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300187/-/DC1.
Communicating editor: S. Wright
Received June 17, 2017.
Accepted August 1, 2017.
Copyright © 2017 Mongue et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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A woman of duty and honour
If you walk through the halls of our communities in the weeks leading up to Remembrance Day you can feel something hanging in the air. It is as if the building's heart is beating with a sense of pride and dedication. Poppies are worn proudly by almost every resident you meet.
If you are luckily enough, you get to sit and chat with someone, it could be a team member or a resident, who has stories to share of a very remarkable life. Ruth Kells is one of those people.
She began her military carer with The Royal Canadian Air Force in January 1944 completing basic training in Ottawa, followed by a six-month course in Montreal as a wireless operator. Working sometimes as a stenographer and others as an operator, Ruth was stationed at posts all across Canada from Nova Scotia to British Columbia. In 1950, Ruth decided to resign and return home to Nova Scotia.
However, Ruth felt a calling during the Korean conflict and decided to return to her military roots as a wireless operator and also joined the RCAF Reserve in Ottawa spending weekends at RCAF Station in Foymount, Ontario. It was there that she was approached to become the Civilian Personnel Officer for the Station and eventually assumed the role of Directorate of Special Radio Activities.
Ottawa didn't just bring Ruth into the RCAF Reserves, it also brought her in contact with a man named Wesley Kells, a Lt. Colonel in the Canadian Army who designed and developed signal equipment. Wesley would visit Ruth at Foymount and they married in 1957 and had a son.
In 1989, after her first entry into the military 45 years previous, Ruth decided to retire. However, she remains a very active member of the Royal Canadian Legion and the Royal Canadian Air Force Association. Ruth was nominated to represent all the Canadian Legions across Canada in Dieppe, France for the 75th Dieppe anniversary, laying wreaths at cenotaphs.
Thank you Ruth for your long standing service and dedication.
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Bethlehem Tilahun Alemu is founder and Managing Director of soleRebels, the world's fastest-growing African footwear brand and the only Fair Trade-certified footwear company in the world. Growing up in a poor suburb of Addis Ababa, Bethlehem decided that there was only one way to defeat poverty – use local craftsmanship to make products that can compete in the global marketplace. She decided on footwear and today 70,000 pairs of shoes leave her factory every year.
soleRebels has been expanding rapidly and has 18 stores around the world, including in Silicon Valley (USA), Japan, Singapore, Austria, Greece, Spain and Switzerland, along with an aggressive e-commerce marketing strategy. It expects to open another 50 to 60 stores in next 18 to 36 months. In 2015, Bethlehem launched an new venture, Republic of Leather, offering bespoke, hand crafted Leather wear and accessories.
Read Bethlehem's full 100 Lionesses profile here.