Flying lemurs – The 'flying tree shrews'? Molecular cytogenetic evidence for a Scandentia-Dermoptera sister clade

Background Flying lemurs or Colugos (order Dermoptera) represent an ancient mammalian lineage that contains only two extant species. Although molecular evidence strongly supports that the orders Dermoptera, Scandentia, Lagomorpha, Rodentia and Primates form a superordinal clade called Supraprimates (or Euarchontoglires), the phylogenetic placement of Dermoptera within Supraprimates remains ambiguous. Results To search for cytogenetic signatures that could help to clarify the evolutionary affinities within this superordinal group, we have established a genome-wide comparative map between human and the Malayan flying lemur (Galeopterus variegatus) by reciprocal chromosome painting using both human and G. variegatus chromosome-specific probes. The 22 human autosomal paints and the X chromosome paint defined 44 homologous segments in the G. variegatus genome. A putative inversion on GVA 11 was revealed by the hybridization patterns of human chromosome probes 16 and 19. Fifteen associations of human chromosome segments (HSA) were detected in the G. variegatus genome: HSA1/3, 1/10, 2/21, 3/21, 4/8, 4/18, 7/15, 7/16, 7/19, 10/16, 12/22 (twice), 14/15, 16/19 (twice). Reverse painting of G. variegatus chromosome-specific paints onto human chromosomes confirmed the above results, and defined the origin of the homologous human chromosomal segments in these associations. In total, G. variegatus paints revealed 49 homologous chromosomal segments in the HSA genome. Conclusion Comparative analysis of our map with published maps from representative species of other placental orders, including Scandentia, Primates, Lagomorpha and Rodentia, suggests a signature rearrangement (HSA2q/21 association) that links Scandentia and Dermoptera to one sister clade. Our results thus provide new evidence for the hypothesis that Scandentia and Dermoptera have a closer phylogenetic relationship to each other than either of them has to Primates.

Cross-species chromosome painting [25,26] is a powerful method for investigating the evolution of genome organizations. This method enables (1) the rapid and reliable identification of homologous chromosome segments between any two species in placental mammals based on DNA sequence homology, (2) the tracking of chromosomal rearrangements which have occurred during evolution based on the distribution pattern of conserved chromosome segments, and (3) an independent verification of the molecular phylogenetic tree using signature chromosomal rearrangements [27][28][29][30][31][32][33][34]. Chromosomal homologies between human and representative species of other placental orders have been established by cross-species chromosome painting using human chromosomespecific probes. Up to now, only two (Dermoptera and Hyracoidea) of the 18 extant placental orders have no published genome-wide comparative maps with human. While data from these orders are critical for understanding the evolution of genome organization of placental mammals as a whole, data from Dermoptera are also pivotal for the reconstruction of the ancestral karyotype and evolutionary history of all primates.
Here, for the first time, we have established the genomewide chromosomal correspondence between human and the Malayan flying lemur (Galeopterus variegatus, GVA, 2n = 56), one of the two living species in Dermoptera, by reciprocal cross-species chromosome painting using human and GVA probes. Comparative analysis of available comparative chromosome maps of representative species of Supraprimates has revealed cytogenetic evidence that unites the Scandentia and Dermoptera into one clade.

Results
The G-banded karyotype and flow karyotype of G. variegatus As reported previously [35], G. variegatus has a 2n = 56 karyotype consisting of eight pairs of bi-armed (# 2, 4-6, 8, 11, 13 and 27) and 19 pairs of acrocentric autosomes ( Figure 1). Chromosome 27 has a secondary constriction, the X chromosome is a large metacentric chromosome, and the Y chromosome is the smallest bi-armed chromosome. The G-banded karyotype of G. variegatus has not been reported previously.
The chromosomes of G. variegatus were resolved into 22 separate regions ( Figure 2). Chromosome paints prepared from individual regions were hybridized to metaphases of G. variegatus, allowing the identification of chromosomes contained in each region. Except for four regions, all other regions each contained only one type of G. variegatus chromosomes. Of these four regions, two regions each contained two G. variegatus chromosomes (GVA8+9 and 16+17), the other two regions each contained three G. variegatus chromosomes (GVA18+19+20 and 24+25+26). A complete set of G. variegatus chromosome-specific paints except for the Y was generated.

Reciprocal painting between G. variegatus and human
To establish the genome-wide chromosomal correspondence between G. variegatus and human, the 22 human autosomal probes and the X probe were firstly hybridized onto G. variegatus chromosomes. To further verify these results, and to define the origin of the homologous human chromosomal segments in the segment associations detected in the G. variegatus genome, we carried out reverse painting of G. variegatus chromosome-specific paints onto human chromosomes. Examples are shown in Figure 3, and the results are summarized against an idiogram of the G-banded HSA karyotype (Figure 4). Eighteen G. variegatus paints (GVA4, 5, 7, 8, 10, 12-19, 23-26 and X) each labeled one HSA chro-G-banded karyotype of G. variegatus (2n = 56, GVA), with a summary of the chromosome painting with human paints mosome or chromosomal segment. Four paints (GVA2, 9, 20 and 27) each hybridized to two HSA chromosomal segments. Three paints (GVA6, 11 and 22) each painted three HSA chromosomal segments. GVA paint 1 gave hybridization signals on four HSA chromosomal segments, and GVA paints 3 and 21 each produced hybridization signals on five HSA chromosomal segments. In total, G. variegatus paints revealed 49 homologous chromosomal segments in the HSA genome.

Discussion
Resolving the phylogenetic placement of Dermoptera within Euarchonta is of fundamental importance to our understanding of the origin and evolution of human genome organization. The establishment of a genomewide comparative chromosome map between human and G. variegatus, filled our knowledge gap on the comparative genome organization of Dermoptera, and ensured that each order contained in the Euarchonta group has at least one representative species for which a genome-wide comparative map with human is available. An integrated analysis of the distribution patterns of synteny-conserved homologous segments in Dermoptera and in the representative species of Primates, Scandentia, Rodentia and Lagomorpha, enabled us to search for signature rearrangements that might provide further insight into the phylogenetic placement of Dermoptera within Euarchonta and Supraprimates.
It has been demonstrated that chromosomal rearrangements (including shared derived chromosomal syntenic associations revealed through chromosome painting) can serve as valuable phylogenetic markers to interpret the relationships of the 18 extant placental orders by the principle of parsimony [27,29,[31][32][33][34][36][37][38][39][40]. When a derived chromosomal association (segments that are syntenic to two or more human chromosomes found on a single chromosome in another species) is shared by various species, this particular chromosome character (that is, a synapomorphy) may reflect a common evolutionary origin or a close phylogenetic relationship of these species [27,28,33]. For instance, the HSA5/21 and 1/19p in Afrotheria [31,32,39], and HSA7q/10p and 2/8 in Xenarthra [40], are considered as signature rearrangements for the superordinal clades Afrotheria and Xenarthra, respectively.
We have compared the above chromosome painting data of different representative species of each order within the Supraprimates. However, we failed to detect any signature rearrangement that would support a close phylogenetic relationship either between G. variegatus and Primates or between Dermoptera and Glires. Although the G. variegatus and the New World monkeys share the one derived association of HSA10/16, while G. variegatus, rabbits and squirrels share the HSA1/10 association, the reverse painting results indicate that the two associations detected in G. variegatus and the other species had different origins. The segments contained in the HSA10/16 association of G.
Summary of the chromosome painting with G. variegatus probes onto human on a G-banded idiogram Figure 4 Summary of the chromosome painting with G. variegatus probes onto human on a G-banded idiogram. Human chromosome numbers are indicated below the chromosomes and the segments homologous to G. variegatus are indicated to the right of each chromosome.
variegatus originated from HSA10p and 16p, while in the New World monkeys, they were derived from HSA10q and 16p [61]. In the genome of G. variegatus, the segments contained in HSA1/10 association are homologous to HSA1q and10q (Figure 4), while in the genomes of rabbits and squirrels, they are homologous to HSA1q and10p [55,60]. Furthermore, comparison of the chromosome map between human and rabbits [55] with the map between human and T. belangeri [53] reveals no shared derived human chromosomal association that unites Scandentia and Lagomorpha into one group either.
The most salient finding comes from the comparison of the comparative chromosome map between human and G. variegatus with the map between human and T. belangeri. Such a comparison revealed one common derived human syntenic segment association, HSA2q/21, in both T. belangeri and G. variegatus. The results of reverse painting using the probes of T. belangeri and G. variegatus to hybridize human chromosomes demonstrated that HSA2q/21 association in both T. belangeri and G. variegatus originated from the same human homologous segments: HSA2q and 21( Figure 4 in this study, and Figure 5 in [53]). This syntenic association has not been detected in any other mammal so far studied by chromosome painting, including bats (order Chiroptera). We thus believe that the HSA2/21 association most likely represents the signature rearrangement that unites Scandentia and Dermoptera orders into one sister clade. Such a clade has been proposed previously by several molecular studies but with moderate to weak bootstrap support [4][5][6][7]22]. This grouping is in sharp contrast to a most recent molecular study that suggested the flying lemur as the closest living relative of primates [21]. It should be mentioned that so far only one representative species from each of Scandentia and Dermoptera has been studied by chromosome painting. Most species in Scandentia have a karyotype 2n = 52-68, with only one species (Urogale everetti) having 2n = 44 [62], while in Dermoptera there is the species (Cynocephalus volans) with 2n = 38 [63]. Further chromosome painting between human and these low diploid number species will help to validate if the HSA2q/21 association is indeed the true signature rearrangement that link Scandentia and Dermoptera into one clade.
In addition to the new insight into the phylogenetic affinity of Dermoptera within Supraprimates, the establishment of the first G-banded karyotype for G. variegatus and the human-G. variegatus comparative map, together with the published human-T. belangeri comparative map, has provided us with an opportunity to compare the Gbanded karyotypes of the T. belangeri and G. variegatus in detail on the basis of genome-wide homologies defined by chromosome painting (Figure 5). Ignoring the morphological changes due to variations in the amount and distribution of heterochromatin, 12 G. variegatus autosomes (GVA1, 2, 7-9, 11, 13, 14, 17, 18, 20 and 26) show one-to-one correspondence with their T. belangeri counterparts; six G. variegatus autosomes (GVA10, 15,19,[23][24][25] each correspond to one T. belangeri autosomal segments; nine G. variegatus autosomes (GVA3-6, 12, 16, 21, 22 and 27) each correspond to between two and four T. belangeri chromosomes or chromosomal segments. The G-banding comparison demonstrates that most homologous chromosomes or chromosomal segments between T. belangeri and G. variegatus defined by comparative painting also show matching G-banding patterns. It is noteworthy that the acrocentric G. variegatus chromosome 1 (= HSA2/21/3), the largest autosome that also carries the HSA2/21 association unique for G. variegatus and T. belangeri, is conserved entirely. In contrast, G. variegatus chromosome 11 (= TBE5) that carried the ancestral placentals syntenic association of HSA16/19 has undergone an inversion as demonstrated by the chromosome painting and banding comparisons. At the current resolution level of G-banding and cross-species chromosome painting, our results suggest that simple chromosomal fissions/ fusions are the predominant mechanism that differentiates the karyotypes of G. variegatus and T. belangeri. However, the conservation in G-banding patterns does not necessarily indicate a close phylogenetic relationship between G. variegatus and T. belangeri. Although we have found only one interchromosomal rearrangement that unites Scandentia and Dermoptera into one clade, small and cryptic intrachromosomal rearrangements (inversions) will escape detection by the current methods. Further comparative high-resolution gene mapping in flying lemurs, tree shrews as well as outgroup species such as the rabbits, squirrels and prosimians may reveal some intrachromosomal rearrangements that could serve as additional cytogenetic evidence for the proposed Scandentia and Dermoptera sister clade.

Conclusion
We have characterized the G-banded karyotype of G. variegatus and established the first genome-wide comparative chromosome map between human and G. variegatus by reciprocal chromosome painting. Comparative analysis of the chromosome painting data from representative species of each order within the superordinal clade Supraprimates provides new molecular cytogenetic evidence that supports a sister-clade relationship between Dermoptera and Scandentia. However, our analysis has failed to identify any cytogenetic signature for well-defined clades such as the Euarchonta and Supraprimates as well as for other controversial clades.