Peter A. Larsen

Natural hybridization generates mammalian lineage with species characteristics

Most diploid species arise from single-species ancestors. Hybrid origins of new species are uncommon (except among polyploids) and are documented infrequently in animals. Examples of natural hybridization leading to speciation in mammals are exceedingly rare. Here, we show a Caribbean species of bat (Artibeus schwartzi) has a nuclear genome derived from two nonsister but congeneric species (A. jamaicensis and A. planirostris) and a mitochondrial genome that is from a third extinct or uncharacterized congener. Artibeus schwartzi is self-sustaining, morphologically distinct, and exists in near geographic isolation of its known parent species. Island effects (i.e., area, reduced habitat variability, and geographic isolation) likely have restricted gene flow from parental species into the Caribbean populations of this hybrid lineage, thus contributing to local adaptation and isolation of this newly produced taxon. We hypothesize differential rates of the development of reproductive isolation within the genus and estimate that 2.5 million years was an insufficient amount of time for the development of postzygotic isolation among the three species that hybridized to produce A. schwartzi. Reticulated evolution thus has resulted in a genomic combination from three evolutionary lineages and a transgressive phenotype that is distinct from all other known species of Artibeus. The data herein further demonstrate the phenomenon of speciation by hybridization in mammals is possible in nature.

Phylogenetics and Phylogeography of the Artibeus jamaicensis Complex Based on Cytochrome-b DNA Sequences

Abstract The phylogenetics and phylogeography of the Jamaican fruit-eating bat (Artibeus jamaicensis) were examined based on analysis of DNA sequence variation in the mitochondrial cytochrome-b gene for 176 individuals representing all 13 subspecies of A. jamaicensis (sensu Simmons 2005). Results document that A. jamaicensis (sensu Simmons 2005) comprises 3 monophyletic assemblages that are separated phylogenetically by the presence of A. obscurus, A. lituratus, and A. amplus. According to the mitochondrial DNA sequence variation, A. jamaicensis, A. schwartzi, and A. planirostris are appropriate species-level names for these lineages. Haplotypes identifiable as A. jamaicensis were absent east of the Andes Mountains in South America; haplotypes of A. schwartzi were documented throughout the Lesser Antilles and from northern Venezuela, and haplotypes of A. planirostris were identified east of the Andes Mountains in South America, north of the Orinoco River in Venezuela, and from the southern Lesser Antilles. Haplotypes of Artibeus jamaicensis, A. schwartzi, and A. planirostris were identified sympatrically on Carriacou, a small island in the southern Lesser Antilles that is ecologically monotypic. The magnitude of genetic divergence separating A. jamaicensis, A. planirostris, and A. schwartzi essentially equals the magnitude of genetic divergence distinguishing A. lituratus, A. obscurus, and A. jamaicensis. Studies of the nuclear genome will be required to understand the biological implications of these patterns in the mitochondrial genome.

Operational criteria for genetically defined species: analysis of the diversification of the small fruit-eating bats, Dermanura (Phyllostomidae: Stenodermatinae)

Species diversity and species limits of the small fruit-eating bats, genus Dermanura (Phyllostomidae: Stenodermatinae) were examined. Estimates of species diversity based on classical morphological criteria (current taxonomy) were compared to diversity estimates based on monophyly and cytochrome-b sequence divergence. The most recent taxonomic list included nine species, whereas the genetic based list contained 11: anderseni, azteca, bogotensis, cinerea, glauca, gnoma, phaeotis, rava, rosenbergi, tolteca, and watsoni, of which three (bogotensis Andersen, rava Miller, and rosenbergi Thomas) have been considered synonyms of cinerea, glauca, phaeotis, and tolteca by previous authors. In addition, we consider incomitata to be a synonym of watsoni. Phylogenetic analyses of mtDNA sequences resolved the interrelationships among taxa and prompted us to re-evaluate some morphological characters that support the distinction of all the recognized taxa, therefore providing a robust estimate of species status. A phylogenetic tree revealed a geographic component to the diversification of Dermanura, including a historical connection between western Andean and Middle American biota. In South America, no species has been recorded from both sides of the Andes Mountains, and at least one clade (glauca, gnoma, and bogotensis) is restricted to the eastern versant of the Andes. Using genetic data (monophyly and genetic distance) to identify species we were able to produce testable genealogical and biogeographic hypotheses to facilitate further studies.

Systematics of Malaysian woolly bats (Vespertilionidae: Kerivoula) inferred from mitochondrial, nuclear, karyotypic, and morphological data

Abstract Examining species boundaries using data from multiple independent sources is an appropriate and robust method to identify genetically isolated evolutionary units. We used 5 data sets—cytochrome b (Cytb), cytochrome c oxidase (COI), amplified fragment length polymorphisms (AFLPs), karyotypes, and morphology—to estimate phylogenetic relationships and species limits within woolly bats, genus Kerivoula, from Southeast Asia. We genetically analyzed 54 specimens of Kerivoula from Malaysia, assigned to 6 of the 10 species currently reported from the country. Phylogenetic analyses of nuclear AFLPs (33 specimens) and mitochondrial DNA sequences from Cytb (51 specimens) and COI (48 specimens) resulted in similar statistically supported species-level clades with minimal change in branching order. Using comparisons of cranial and dental morphology and original species descriptions, we assigned the resulting phylogenetic clades to K. hardwickii, K. intermedia, K. lenis, K. minuta, K. papillosa, K pellucida, and 1 unidentified species. Karyotypes further documented variability among the 6 clades. Five different karyotypes were identified, with 2 species having indistinguishable karyotypes. We compared our COI gene sequence data to 110 specimens of Kerivoula from Southeast Asia made available by researchers of the Barcode of Life Database. Our Cytb and AFLP species identifications were congruent with those in the COI database. Intraspecific geographic variation of about 5–7% sequence divergence was observed in Cytb and COI genes within both K. hardwickii and K. minuta. Relaxed molecular clock analyses indicated a late Oligocene to early Miocene origin of the Kerivoulinae with intraspecific diversification events coinciding with the late Pliocene and Pleistocene epochs in at least 6 species. Specimens from Sabah (northeastern Borneo) showed relatively high genetic divergence compared to those between Peninsular Malaysia and Sarawak (southwestern Borneo), indicating Pleistocene or Pliocene refugia in Borneo. We conclude that at least 1 distinct lineage of Kerivoula is yet to be described from Borneo and that the intraspecific geographic variation in some species agrees with previous studies on the diversification of flora and fauna in Borneo.

SPECIATION WITHIN BONNETED BATS (GENUS EUMOPS): THE COMPLEXITY OF MORPHOLOGICAL, MITOCHONDRIAL, AND NUCLEAR DATA SETS IN SYSTEMATICS

Abstract We phylogenetically analyze 705 base pairs of the cytochrome-b gene and 351 amplified fragment length polymorphism (AFLP) bands from populations of the karyotypically variable Wagners bonneted bat, Eumops glaucinus, and the Florida bonneted bat, Eumops floridanus (Chiroptera: Molossidae). Three karyotypes have been documented across the range of E. glaucinus, and we report that the karyotype from Cuba is morphologically similar to that from Jamaica. A 4th karyotype is present in specimens from western Ecuador. Three distinct lineages are present in both the cytochrome-b and AFLP trees. One lineage is restricted to western Ecuador and exhibits cytochrome-b divergence values comparable to the values seen between recognized species of Eumops, suggesting that this lineage represents a distinct species. The other 2 lineages are distributed in disjunct areas: Paraguay and Venezuela; and Mexico, the Caribbean, and the United States. Specimens of E. floridanus are morphologically distinct from E. glaucinus, but cannot be distinguished by examination of cytochrome-b or AFLP DNA data. We conclude that there are 4 species in the E. glaucinus complex—E. glaucinus (South America east of the Andes), E. ferox (Caribbean, Mexico, and Central America), E. floridanus in south Florida, and an unnamed taxon in western Ecuador. Speciation is a complex process and no single mechanism, model, concept, or definition is likely to cover all the diverse patterns observed.

Genetic Diversity of Neotropical Myotis (Chiroptera: Vespertilionidae) with an Emphasis on South American Species

Background Cryptic morphological variation in the Chiropteran genus Myotis limits the understanding of species boundaries and species richness within the genus. Several authors have suggested that it is likely there are unrecognized species-level lineages of Myotis in the Neotropics. This study provides an assessment of the diversity in New World Myotis by analyzing cytochrome-b gene variation from an expansive sample ranging throughout North, Central, and South America. We provide baseline genetic data for researchers investigating phylogeographic and phylogenetic patterns of Myotis in these regions, with an emphasis on South America. Methodology and Principal Findings Cytochrome-b sequences were generated and phylogenetically analyzed from 215 specimens, providing DNA sequence data for the most species of New World Myotis to date. Based on genetic data in our sample, and on comparisons with available DNA sequence data from GenBank, we estimate the number of species-level genetic lineages in South America alone to be at least 18, rather than the 15 species currently recognized. Conclusions Our findings provide evidence that the perception of lower species richness in South American Myotis is largely due to a combination of cryptic morphological variation and insufficient sampling coverage in genetic-based systematic studies. A more accurate assessment of the level of diversity and species richness in New World Myotis is not only helpful for delimiting species boundaries, but also for understanding evolutionary processes within this globally distributed bat genus.

Geogenetic patterns in mouse lemurs (genus Microcebus) reveal the ghosts of Madagascar's forests past

Phylogeographic analysis can be described as the study of the geological and climatological processes that have produced contemporary geographic distributions of populations and species. Here, we attempt to understand how the dynamic process of landscape change on Madagascar has shaped the distribution of a targeted clade of mouse lemurs (genus Microcebus) and, conversely, how phylogenetic and population genetic patterns in these small primates can reciprocally advance our understanding of Madagascars prehuman environment. The degree to which human activity has impacted the natural plant communities of Madagascar is of critical and enduring interest. Today, the eastern rainforests are separated from the dry deciduous forests of the west by a large expanse of presumed anthropogenic grassland savanna, dominated by the Family Poaceae, that blankets most of the Central Highlands. Although there is firm consensus that anthropogenic activities have transformed the original vegetation through agricultural and pastoral practices, the degree to which closed-canopy forest extended from the east to the west remains debated. Phylogenetic and population genetic patterns in a five-species clade of mouse lemurs suggest that longitudinal dispersal across the island was readily achieved throughout the Pleistocene, apparently ending at ∼55 ka. By examining patterns of both inter- and intraspecific genetic diversity in mouse lemur species found in the eastern, western, and Central Highland zones, we conclude that the natural environment of the Central Highlands would have been mosaic, consisting of a matrix of wooded savanna that formed a transitional zone between the extremes of humid eastern and dry western forest types.

New Species of Bonneted Bat, Genus Eumops (Chiroptera: Molossidae) from the Lowlands of Western Ecuador and Peru

We describe and formally name a species of bonneted bat (genus Eumops), which is a member of the E. glaucinus complex. Closely related species are E. glaucinus, E. ferox, and E. floridanus. The conceptual basis for the description of this species is the Genetic Species Concept with speciation by the Bateson-Dobzhanzky-Muller model. The new species is distinguished from all other species of bats by its unique karyotype (2N = 38, FN = 54), sequence of the mitochondrial cytochrome-b gene, and genetic markers revealed through analysis of Amplified Fragment Length Polymorphisms. The series from the type locality (Ecuador, Guayas) is comprised of seven specimens. Morphologically, the new species is smaller than E. floridanus and E. glaucinus, but is indistinguishable from E. ferox. The new species is significantly smaller in size than E. glaucinus in six out of eight measurements and is distinguishable from E. glaucinus based on length of maxillary toothrow and zygomatic breadth. The geographic range of E. wilsoni, as currently documented, is the dry forests of southwestern Ecuador and adjacent northwestern Peru. We propose the common name for this species be Wilsons bonneted bat.

The utility of PacBio circular consensus sequencing for characterizing complex gene families in non-model organisms

BackgroundMolecular characterization of highly diverse gene families can be time consuming, expensive, and difficult, especially when considering the potential for relatively large numbers of paralogs and/or pseudogenes. Here we investigate the utility of Pacific Biosciences single molecule real-time (SMRT) circular consensus sequencing (CCS) as an alternative to traditional cloning and Sanger sequencing PCR amplicons for gene family characterization. We target vomeronasal gene receptors, one of the most diverse gene families in mammals, with the goal of better understanding intra-specific V1R diversity of the gray mouse lemur (Microcebus murinus). Our study compares intragenomic variation for two V1R subfamilies found in the mouse lemur. Specifically, we compare gene copy variation within and between two individuals of M. murinus as characterized by different methods for nucleotide sequencing. By including the same individual animal from which the M. murinus draft genome was derived, we are able to cross-validate gene copy estimates from Sanger sequencing versus CCS methods.ResultsWe generated 34,088 high quality circular consensus sequences of two diverse V1R subfamilies (here referred to as V1RI and V1RIX) from two individuals of Microcebus murinus. Using a minimum threshold of 7× coverage, we recovered approximately 90% of V1RI sequences previously identified in the draft M. murinus genome (59% being identical at all nucleotide positions). When low coverage sequences were considered (i.e. < 7× coverage) 100% of V1RI sequences identified in the draft genome were recovered. At least 13 putatively novel V1R loci were also identified using CCS technology.ConclusionsRecent upgrades to the Pacific Biosciences RS instrument have improved the CCS technology and offer an alternative to traditional sequencing approaches. Our results suggest that the Microcebus murinus V1R repertoire has been underestimated in the draft genome. In addition to providing an improved understanding of V1R diversity in the mouse lemur, this study demonstrates the utility of CCS technology for characterizing complex regions of the genome. We anticipate that long-read sequencing technologies such as PacBio SMRT will allow for the assembly of multigene family clusters and serve to more accurately characterize patterns of gene copy variation in large gene families, thus revealing novel micro-evolutionary patterns within non-model organisms.

Application of circular consensus sequencing and network analysis to characterize the bovine IgG repertoire

BackgroundVertebrate immune systems generate diverse repertoires of antibodies capable of mediating response to a variety of antigens. Next generation sequencing methods provide unique approaches to a number of immuno-based research areas including antibody discovery and engineering, disease surveillance, and host immune response to vaccines. In particular, single-molecule circular consensus sequencing permits the sequencing of antibody repertoires at previously unattainable depths of coverage and accuracy. We approached the bovine immunoglobulin G (IgG) repertoire with the objective of characterizing diversity of expressed IgG transcripts. Here we present single-molecule real-time sequencing data of expressed IgG heavy-chain repertoires of four individual cattle. We describe the diversity observed within antigen binding regions and visualize this diversity using a network-based approach.ResultsWe generated 49,945 high quality cDNA sequences, each spanning the entire IgG variable region from four Bos taurus calves. From these sequences we identified 49,521 antigen binding regions using the automated Paratome web server. Approximately 9% of all unique complementarity determining 2 (CDR2) sequences were of variable lengths. A bimodal distribution of unique CDR3 sequence lengths was observed, with common lengths of 5–6 and 21–25 amino acids. The average number of cysteine residues in CDR3s increased with CDR3 length and we observed that cysteine residues were centrally located in CDR3s. We identified 19 extremely long CDR3 sequences (up to 62 amino acids in length) within IgG transcripts. Network analyses revealed distinct patterns among the expressed IgG antigen binding repertoires of the examined individuals.ConclusionsWe utilized circular consensus sequencing technology to provide baseline data of the expressed bovine IgG repertoire that can be used for future studies important to livestock research. Somatic mutation resulting in base insertions and deletions in CDR2 further diversifies the bovine antibody repertoire. In contrast to previous studies, our data indicate that unusually long CDR3 sequences are not unique to IgM antibodies in cattle. Centrally located cysteine residues in bovine CDR3s provide further evidence that disulfide bond formation is likely of structural importance. We hypothesize that network or cluster-based analyses of expressed antibody repertoires from controlled challenge experiments will help identify novel natural antigen binding solutions to specific pathogens of interest.