Gomes et al. BMC Evolutionary Biology (2018) 18:62 https://doi.org/10.1186/s12862-018-1176-3 RESEARCHARTICLE Open Access Chromosomal evolution and phylogeny in the Nullicauda group (Chiroptera, Phyllostomidae): evidence from multidirectional chromosome painting Anderson José Baia Gomes1,3, Cleusa Yoshiko Nagamachi1,4, Luis Reginaldo Ribeiro Rodrigues2, Malcolm Andrew Ferguson-Smith5, Fengtang Yang6, Patricia Caroline Mary O’Brien5 and Julio Cesar Pieczarka1,4* Abstract Background: The family Phyllostomidae (Chiroptera) shows wide morphological, molecular and cytogenetic variation; many disagreements regarding its phylogeny and taxonomy remains to be resolved. In this study, we use chromosome painting with whole chromosome probes from the Phyllostomidae Phyllostomus hastatus and Carollia brevicauda to determine the rearrangements among several genera of the Nullicauda group (subfamilies Gliphonycterinae, Carolliinae, Rhinophyllinae and Stenodermatinae). Results: These data, when compared with previously published chromosome homology maps, allow the construction of a phylogeny comparable to those previously obtained by morphological and molecular analysis. Our phylogeny is largely in agreement with that proposed with molecular data, both on relationships between the subfamilies and among genera; it confirms, for instance, that Carollia and Rhinophylla, previously considered as part of the same subfamily are, in fact, distant genera. Conclusions: The occurrence of the karyotype considered ancestral for this family in several different branches suggests that the diversification of Phyllostomidae into many subfamilies has occurred in a short period of time. Finally, the comparison with published maps using human whole chromosome probes allows us to track some syntenic associations prior to the emergence of this family. Keywords: Chromosome phylogeny, Molecular cytogenetics, Bat evolution, Genome mapping Background Traditionally, the subfamily Carolliinae consists of The family Phyllostomidae is the third most speciose genera Carollia (10 spp.) and Rhinophylla (3 spp.) within the order Chiroptera, with 60 genera and 200 [3, 4, 6, 7]. However, in the classification proposed species [1], being grouped into 11 subfamilies: Macroti- by Baker et al. [2] these two genera are not closely nae, Micronycterinae, Desmodontinae, Lonchorhininae, related. The subfamily Carolliinae (composed by the Phyllostominae, Glossophaginae, Lonchophyllinae, Car- genus Carollia) was included as sister group to Gly- olliinae, Glyphonycterinae, Rhinophyllinae, and Steno- phonycterinae, a new subfamily that includes Gly- dermatinae [2]. This family shows huge morphological, phonycteris and Trinycteris that were previously molecular and cytogenetic variation, both between and subgenera within Micronycteris [8–10], and later within species, in addition to much controversy about were raised to the genus level [3, 11]. Rhinophylla its taxonomy [2–5]. was included in its own subfamily (Rhinophyllinae) that was seen as closely related to the subfamily * Correspondence: [email protected] Stenodermatinae. 1Laboratório de Citogenética, CEABIO, ICB, Universidade Federal do Pará, Av. Bernardo Sayão, sn. Guamá, Belém, Pará 66075-900, Brazil In the Baker et al. [2] classification, the group of Car- 4CNPQ Researcher, Brasilia, Brazil olliinae + Glyphonycterinae + Rhinophyllinae + Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is 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 you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Gomes et al. BMC Evolutionary Biology (2018) 18:62 Page 2 of 10 Stenodermatinae was designated Nullicauda, an un- Methods ranked taxon, initially proposed by Wetterer et al. [3] for Specimens examined the group consisting of subfamilies Carolliinae and Chromosome banding and painting were used to analyze Stenodermatinae. the karyotypes of bats from three phyllostomid subfam- Cytogenetic studies in species of the Nullicauda ilies, Glossophaginae, Glyphonycterinae and Rhinophylli- group show extensive interspecific and intraspecific nae (Table 1), with the last two being members of the chromosome variation [12]. To further investigate the Nullicauda group. The specimens were captured with controversial phylogenetic hypotheses based on mor- mist nets. Chromosome preparations and tissue biopsies phological and molecular data in Nullicauda, we ana- were sent to the Cytogenetic Laboratory of the Federal lyzed chromosome evolution in this group through University of Pará. Specimens were deposited in the multidirectional chromosome painting using whole Mastozoology collection of the Emilio Goeldi Museum chromosome paint probes from two phyllostomid and Zoology Museum of the Western Pará University. bats, Phyllostomus hastatus (PHA) and Carollia brevi- cauda (CBR) [13], in the species Rhinophylla pumilio, Chromosome preparations, cell culture and chromosome Rhinophylla aff. fischerae, Trinycteris nicefori and banding Glossophaga soricina. The results obtained here were Metaphase chromosomes were obtained from direct compared with those previously published with the bone marrow extraction [16], and through primary same probes in other species, in order to construct a culture of fibroblasts [17]. G-banding patterns were phylogeny based on chromosomal homology data. We obtained from incubation in enzymatic trypsin solu- also integrated the comparative chromosome maps tion [18], and subsequent incubation in saline solu- obtained with these probes with maps generated using tion (0.5X SSC) and Wright dye staining diluted in human probes in Glossophaga soricina [14, 15]. phosphate buffer at a ratio of 1: 3 v / v. Staining time Table 1 Species and karyotypes analyzed in the present work and data from literature used on mapping comparison Scientific Names and Subfamilies 2 N FN Locality Number and Cross-species Abbreviations sex FISH Rhinophylla aff. fischeraea, Rhinophyllinae 38 68 Juruti (2°08′40”S; 56°05′28”W) and Santarém (2°26″54”S; 3 (1 M, 2F) This study RFI 54°42″07”W) Rhinophylla pumilio, RPU Rhinophyllinae 34 62 Santa Barbara (1°13′31”S; 48°17″51”W) 3 (2 M, 1F) This study Glossophaga soricina, GSO Glossophaginae 32 60 Belém (1°28′05”S; 48°26′35”W) 1 (1F) This study Trinycteris nicefori, TNI Glyphonycterinae 28 52 Oriximiná (1°45′40”S; 55°51′52”W) and Santarém (2°26″ 3 (1 M, 2F) This study 54”S; 54°42″07”W) Phyllostomus hastatus, PHA Phyllostominae 32 60 Pieczarka et al. 2005 Carollia brevicauda, CBR Carolliinae 20/ 36 Pieczarka et al. 21 2005 Artibeus obscurus, AOB Stenodermatinae 30/ 56 Pieczarka et al. 31 2013 Uroderma magnirostrum, Stenodermatinae 36 62 Pieczarka et al. UMA 2013 Uroderma bilobatum, UBI Stenodermatinae 42 50 Pieczarka et al. 2013 Chiroderma villosum, CVI Stenodermatinaeb 26 48 Gomes et al. 2016 Mesophylla macconnelli, Stenodermatinaeb 21/ 18 Gomes et al. MMA 22 2016 Vampyressa thyone, VTH Stenodermatinaeb 23/ 20 Gomes et al. 24 2016 Vampyriscus bidens, VBI Stenodermatinaeb 26 48 Gomes et al. 2016 Vampyriscus brocki, VBR Stenodermatinaeb 24 44 Gomes et al. 2016 aThe species here is called aff.(=affinis) because Gomes et al. (2010) demonstrated that, despite morphologically similar to Rhinophylla fischerae, its karyotype diverges clearly from the one described previously for that species by Baker and Bleier (1971) in a sample from Colombia. However, further studies are necessary to formally confirm that it is a different species. bSubtribe Vampyressina. M = male; F = female Gomes et al. BMC Evolutionary Biology (2018) 18:62 Page 3 of 10 was 5 min. Karyotypes were organized following the its karyotype is similar to Macrotus californicus pattern in the literature for each species. (MCA), a species of the most basal genus of Phyllos- tomidae according to Baker et al. [2]; and 3) its Fluorescence in situ hybridization (FISH) karyotype is similar to the ancestral karyotype [22]. Multidirectional chromosome painting was performed The analysis of Maximum Parsimony was made using using whole chromosome probes from CBR and PHA PAUP 4.0b10 (Phylogenetic Analysis Using Parsi- (Phyllostomidae), generated by flow cytometry [13]. The mony) [23]. A heuristic search to find the most par- probes were amplified and labeled by DOP-PCR [19, 20] simonious tree(s) was performed using Tree Bisection and hybridized following procedures previously de- Reconnection (TBR) branch-swapping; the posterior scribed [13, 20]. Briefly, the slides were incubated in a bootstrap probability was obtained with one thousand pepsin enzyme solution, washed in 2× SSC and dehy- replicates. drated in an alcohol series (70%, 90% and 100%). Subse- quently the slides were oven aged at 65 °C for 1 hour, Results denatured for 1 min in formamide solution (70% form- Rhinophylla pumilio amide/ 2× SSC) and incubated in hybridization solution Rhinophylla pumilio (RPU) shows a karyotype with (14 μl of solution containing: 50% formamide, 2× SSC, 2n = 34, and FN = 62 (Fig. 1a), as well as G-, C- 10% dextran sulfate, 5 μg of salmon sperm DNA, 2 μg banding and Ag-NOR staining,