Anonymous Nuclear Loci Research Articles

High vagility facilitates population persistence and expansion prior to the Last Glacial Maximum in an antarctic top predator: The Snow petrel (Pagodroma nivea)

AbstractAimsPleistocene glacial cycles have had profound effects on the distribution and genetic diversity of high latitude species, which can vary with species‐specific traits, such as vagility. Demographic responses of antarctic flying seabirds to the same events remain unassessed. We addressed this knowledge gap by studying the genetic population connectivity and demographic history of a flying seabird endemic to Antarctica, the Snow petrel. We hypothesize that their high vagility due to flight may represent an advantage over non‐flying seabirds in enduring past climate variation.LocationApproximately 3,000 km of coastline in East Antarctica, covering three areas in Mac. Robertson Land, Princess Elizabeth Land and Wilkes Land. An inland location was also sampled at the Prince Charles Mountains, Mac. Robertson Land.TaxonSnow petrel (Pagodroma nivea).MethodsWe sampled 93 individuals and sequenced a total of 5,412 base pairs, including two mitochondrial genes, four anonymous nuclear loci and a nuclear intron. We used frequentist and Bayesian approaches to examine population genetic structuring and an Extended Bayesian Skyline Plot method to infer the demographic history of the species in the study area. In addition, evidence of exposed bedrock during glacial periods was summarized in maps of the studied area representing potential refugia for the species.ResultsDifferentiation indexes, genetic clustering and haplotype networks suggest long‐term population connectivity for Snow petrels across the study area, with no evidence for reliction into refugia that were genetically isolated. Significantly, population expansions pre‐dated the Last Glacial Maximum (LGM), but only where there was evidence of ice‐free areas during this period.Main conclusionsThe high vagility of Snow petrels may have been advantageous for access to foraging areas and supported large populations despite the harsh conditions during the LGM. Our results highlight that species‐specific traits can exert a strong influence on demographic responses to the same environmental events.

Plio-Pleistocene diversification and biogeographic barriers in southern Australia reflected in the phylogeography of a widespread and common lizard species

Palaeoclimatic events and biogeographical processes since the mid-Tertiary have played an important role in shaping the evolution and distribution of Australian fauna. However, their impacts on fauna in southern and arid zone regions of Australia are not well understood. Here we investigate the phylogeography of an Australian scincid lizard, Tiliqua rugosa, across southern Australia using mitochondrial DNA (mtDNA) and 11 nuclear DNA markers (nuDNA), including nine anonymous nuclear loci. Phylogenetic analyses revealed three major mtDNA lineages within T. rugosa, geographically localised north and south of the Murray River in southern Australia, and west of the Nullarbor Plain. Molecular variance and population analyses of both mtDNA and nuDNA haplotypes revealed significant variation among the three populations, although potential introgression of nuDNA markers was also detected for the Northern and Southern population. Coalescent times for major mtDNA lineages coincide with an aridification phase, which commenced after the early Pliocene and increased in intensity during the Late Pliocene-Pleistocene. Species distribution modelling and a phylogeographic diffusion model suggest that the range of T. rugosa may have contracted during the Last Glacial Maximum and the locations of optimal habitat appear to coincide with the geographic origin of several distinct mtDNA lineages. Overall, our analyses suggest that Plio-Pleistocene climatic changes and biogeographic barriers associated with the Nullarbor Plain and Murray River have played a key role in shaping the present-day distribution of genetic diversity in T. rugosa and many additional ground-dwelling animals distributed across southern Australia.

Looking for morphological evidence of cryptic species in Asterina Nardo, 1834 (Echinodermata: Asteroidea). The redescription of Asterina pancerii (Gasco, 1870) and the description of two new species

Three species of the genus Asterina are known to inhabit the Mediterranean Sea and the north-eastern Atlantic Ocean: Asterina gibbosa (Pennant, 1777), A. pancerii (Gasco, 1870) and A. phylactica Emson & Crump, 1979. Differentiation of these species has primarily been based only on subtle characters (some highly debatable), such as colour or size. Therefore, this study aimed to review the morphological data characterising members of the genus, to incorporate new characters that may clarify morphological analyses and to couple morphological data with molecular evidence of differentiation based on the analysis of partial sequences of the cytochromec oxidase subunitI (COI) and 18S rDNA (18S) genes and two anonymous nuclear loci (AgX2 and AgX5). The different lineages and cryptic species identified from the molecular analysis were then morphologically characterised, which was challenging given the limited number of diagnostic characters. Two of the five monophyletic lineages obtained molecularly (COI divergence >4%), further supported by differences in morphological characters and reproductive behaviour, are proposed as new species: Asterina martinbarriosi, sp. nov. from the Canary Islands, Spain (eastern central Atlantic Ocean) and Asterina vicentae, sp. nov. from Tarragona, north-eastern Spain (western Mediterranean Sea).

Reassessment of a possible case of intraspecific gene flow across Australia’s Great Dividing Range in the variegated fairy wren, Malurus lamberti (Aves: Maluridae), and its systematic consequences

Two subspecies of the variegated fairy wren Malurus lamberti, Malurus lamberti lamberti and Malurus lamberti assimilis, are thought to exemplify a surprisingly rare case of intraspecific gene flow across eastern Australia's Great Dividing Range. We screened 71 individuals within and beyond their putative hybrid zone for diversity in one mitochondrial and two anonymous nuclear loci, four microsatellite markers and diagnostically different plumage traits. Almost all supposed hybrid zone individuals were genotypically and phenotypically M. l. assimilis, but some carried mitochondrial DNA typical of M. l. lamberti. We infer an intermittently 'leaky' genetic boundary between the two taxa. Integrating this study with our earlier work on the M. lamberti group, we argue that speciation, albeit with some gene flow, between M. l. lamberti and all other taxa recently assigned to M. lamberti is essentially complete. A two-species taxonomy whereby one species, M. lamberti Vigors and Horsfield, 1827 (variegated fairy wren), comprises only what is currently called M. l. lamberti, and a second species, Malurus assimilis North, 1901 (purple-backed fairy wren), is for all other populations recently assigned to M. lamberti should be adopted and further tested.

Development of Anonymous Nuclear Loci for Pterospora andromedea (Monotropoideae) Using Illumina and Ion Torrent Sequencing Data

The mycoheterotrophic plant Pterospora andromedea is an important species in understanding the evolution and ecology of highly specialized plant-fungal symbioses. Lineages within P. andromedea have been shown to engage in highly specific interactions with obligate fungal associates found in Rhizopogon subgenus Amylopogon. Unfortunately, evidence of highly specific interactions resulted from single locus analyses and this result requires further examination. If highly specific host-symbiont selectivity is present, then the lack of compatible partners may best explain the conservation status of P. andromedea. To better evaluate and support conservation efforts for P. andromedea, more molecular loci are necessary. 115 candidate loci were screened from Illumnia MiSeq and Ion Torrent next-generation sequencing and resulted in the development of four polymorphic anonymous nuclear loci. These loci will provide the means to critically examine host-symbiont specificity for P. andromedea lineages across the endemic range and will assist conservation initiatives.

Increased phylogenetic resolution within the ecologically important Rhizopogon subgenus Amylopogon using 10 anonymous nuclear loci

ABSTRACTRhizopogon species are ecologically significant ectomycorrhizal fungi in conifer ecosystems. The importance of this system merits the development and utilization of a more robust set of molecular markers specifically designed to evaluate their evolutionary ecology. Anonymous nuclear loci (ANL) were developed for R. subgenus Amylopogon. Members of this subgenus occur throughout the United States and are exclusive fungal symbionts associated with Pterospora andromedea, a threatened mycoheterotrophic plant endemic to disjunct eastern and western regions of North America. Candidate ANL were developed from 454 shotgun pyrosequencing and assessed for positive amplification across targeted species, sequencing success, and recovery of phylogenetically informative sites. Ten ANL were successfully developed and were subsequently used to sequence representative taxa, herbaria holotype and paratype specimens in R. subgenus Amylopogon. Phylogenetic reconstructions were performed on individual and concatenated data sets by Bayesian inference and maximum likelihood methods. Phylogenetic analyses of these 10 ANL were compared with a phylogeny traditionally constructed using the universal fungal barcode nuc rDNA ITS1-5.8S-ITS2 region (ITS). The resulting ANL phylogeny was consistent with most of the species designations delineated by ITS. However, the ANL phylogeny provided much greater phylogenetic resolution, yielding new evidence for cryptic species within previously defined species of R. subgenus Amylopogon. Additionally, the rooted ANL phylogeny provided an alternate topology to the ITS phylogeny, which inferred a novel set of evolutionary relationships not identified in prior phylogenetic studies.

Dams impact westslope cutthroat trout metapopulation structure and hybridization dynamics

Dams fragment aquatic networks and impact the evolutionary dynamics of migratory fish populations. Historically, the Lake Pend Oreille–lower Clark Fork River system in Idaho and Montana supported a large metapopulation of adfluvial westslope cutthroat trout (westslope). These fish matured in the lake and made upstream spawning migrations to their natal tributaries in the river. Currently, dams allow downstream passage for juvenile fish to the lake, but no upstream passage for adults. We examined 19 microsatellite and anonymous nuclear loci to characterize spatial patterns of non-native rainbow trout (rainbow) genetic introgression in westslope populations upstream and downstream of these dams. We detected hybridization at 20 of 42 sites sampled throughout the system, with rainbow introgression ranging from 1.1 to 99.8%. Rainbow introgression was higher in tributaries downstream of both dams (12 of 18 sites downstream vs 6 of 22 sites upstream). Within tributaries, hybrids were more common in lower sections and only one sampled site contained a hybrid swarm. Fish collected below both dams during the spawning migration were admixtures of westslope, rainbows and hybrids with an average rainbow introgression of 48%. Phenotypic criteria reduced rainbow introgression to less than 1% for putative westslope identified for selective upstream transport at both dams. Lack of upstream fish passage at these dams has likely constrained spread of hybridization and increased isolation among non-hybridized westslope populations by reducing the abundance of the migratory life history form in areas upstream of these dams.

Mechanisms of global diversification in the marine species Madeiran Storm-petrel Oceanodroma castro and Monteiro’s Storm-petrel O. monteiroi: Insights from a multi-locus approach

The evolutionary mechanisms underlying the geographic distribution of gene lineages in the marine environment are not as well understood as those affecting terrestrial groups. The continuous nature of the pelagic marine environment may limit opportunities for divergence to occur and lineages to spatially segregate, particularly in highly mobile species. Here, we studied the phylogeography and historical demography of two tropically distributed, pelagic seabirds, the Madeiran Storm-petrel Oceanodroma castro, sampled in the Azores, Madeira, Galapagos and Japan, and its sister species Monteiro’s Storm-petrel O. monteiroi (endemic to the Azores), using a multi-locus dataset consisting of 12 anonymous nuclear loci and the mitochondrial locus control region. Both marker types support the existence of four significantly differentiated genetic clusters, including the sampled O. monteiroi population and three populations within O. castro, although only the mitochondrial locus suggests complete lineage sorting. Multi-locus coalescent analyses suggest that most divergence events occurred within the last 200,000years. The proximity in divergence times precluded robust inferences of the species tree, in particular of the evolutionary relationships of the Pacific populations. Despite the great potential for dispersal, divergence among populations apparently proceeded in the absence of gene flow, emphasizing the effect of non-physical barriers, such as those driven by the paleo-oceanographical environments, philopatry and local adaptation, as important mechanisms of population divergence and speciation in highly mobile marine species. In view of the predicted climate change impacts, future changes in the demography and evolutionary dynamics of marine populations might be expected.

Molecular Phylogeny of the Lizard Clade Leiosaurae Endemic to Southern South America

Abstract: The clade Leiosaurae currently includes 18 species in the genera Diplolaemus, Leiosaurus, and Pristidactylus. It is mainly distributed in Argentina from 30° latitude south in the northwestern region of the country, to 52° south in Patagonia, from 63° longitude in coastal areas to 73° along the Andean Cordillera, across multiple habitats and including a small area in Chile. Several morphological and molecular taxonomic studies on a subset of these species have been published, but no comprehensive phylogenetic hypothesis is available for the clade. The objective of this work is to present a molecular phylogenetic hypothesis for the majority of the described species in the clade. We sequenced two mitochondrial genes, five nuclear protein coding, and three anonymous nuclear loci, and implemented traditional concatenated analyses as well as a species-tree approach. All methods inferred very similar topologies. We found the genera Diplolaemus and Leiosaurus to be monophyletic, whereas P. torquatus wa...

Anonymous nuclear markers reveal taxonomic incongruence and long-term disjunction in a cactus species complex with continental-island distribution in South America

The Pilosocereus aurisetus complex consists of eight cactus species with a fragmented distribution associated to xeric enclaves within the Cerrado biome in eastern South America. The phylogeny of these species is incompletely resolved, and this instability complicates evolutionary analyses. Previous analyses based on both plastid and microsatellite markers suggested that this complex contained species with inherent phylogeographic structure, which was attributed to recent diversification and recurring range shifts. However, limitations of the molecular markers used in these analyses prevented some questions from being properly addressed. In order to better understand the relationship among these species and make a preliminary assessment of the genetic structure within them, we developed anonymous nuclear loci from pyrosequencing data of 40 individuals from four species in the P. aurisetus complex. The data obtained from these loci were used to identify genetic clusters within species, and to investigate the phylogenetic relationship among these inferred clusters using a species tree methodology. Coupled with a palaeodistributional modelling, our results reveal a deep phylogenetic and climatic disjunction between two geographic lineages. Our results highlight the importance of sampling more regions from the genome to gain better insights on the evolution of species with an intricate evolutionary history. The methodology used here provides a feasible approach to develop numerous genealogical molecular markers throughout the genome for non-model species. These data provide a more robust hypothesis for the relationship among the lineages of the P. aurisetus complex.

Phylogeographic inference using Bayesian model comparison across a fragmented chorus frog species complex.

Fragmented species complexes provide an interesting system for investigating biogeographic history and the present distribution of genetic variation. Recent advances in sequencing technology and statistical phylogeography enable the collection and rigorous analysis of large multilocus data sets, but designing studies that produce meaningful phylogeographic inferences remains challenging. We implemented a Bayesian model comparison approach to investigate previous biogeographic hypotheses while simultaneously inferring the presence of genetic structure in a chorus frog species complex. The Illinois chorus frog (Pseudacris illinoensis), originally described as a subspecies of the broadly distributed Strecker's chorus frog (Pseudacris streckeri), occurs in small, disjunct regions associated with scarce sand prairie habitats that have been impacted by human development. We used high-throughput sequencing to develop and collect a multitiered genetic data set comprised of three different marker types (23 anonymous nuclear sequence loci, four mitochondrial genes and 14 microsatellite loci) designed to address questions across different evolutionary timescales. Phylogenetic analyses uncovered a deep divergence between populations in the Edwards Plateau of central Texas and all other P.streckeri/P. illinoensis populations, but suggest the disjunct distribution of P.illinoensis occurred more recently. Our best-supported migration model is consistent with the hypothesis that central Texas represented a refugium from which populations expanded via multiple routes. This model also indicates that disjunct northern and southern regions of P.illinoensis should be considered genetically distinct management units. Our study provides an evolutionary context for future studies and conservation efforts in P.illinoensis and demonstrates the utility of model-based approaches for phylogeographic inference.

King penguin demography since the last glaciation inferred from genome-wide data.

How natural climate cycles, such as past glacial/interglacial patterns, have shaped species distributions at the high-latitude regions of the Southern Hemisphere is still largely unclear. Here, we show how the post-glacial warming following the Last Glacial Maximum (ca 18 000 years ago), allowed the (re)colonization of the fragmented sub-Antarctic habitat by an upper-level marine predator, the king penguin Aptenodytes patagonicus. Using restriction site-associated DNA sequencing and standard mitochondrial data, we tested the behaviour of subsets of anonymous nuclear loci in inferring past demography through coalescent-based and allele frequency spectrum analyses. Our results show that the king penguin population breeding on Crozet archipelago steeply increased in size, closely following the Holocene warming recorded in the Epica Dome C ice core. The following population growth can be explained by a threshold model in which the ecological requirements of this species (year-round ice-free habitat for breeding and access to a major source of food such as the Antarctic Polar Front) were met on Crozet soon after the Pleistocene/Holocene climatic transition.

Multilocus phylogeny of the widely distributed South American lizard clade Eulaemus (Liolaemini, Liolaemus)

The lizard genus Liolaemus and different clades within it have been the focus of several recent phylogenetic studies mainly based on morphology and mtDNA. Although there is general consensus for recognizing two clades (subgenera) within the genus, [Liolaemus (sensu stricto) and Eulaemus], phylogenetic relationships within each subgenus remain difficult to elucidate, given incomplete taxonomic sampling and large discordance between published studies. Here, new phylogenetic relationships for the Eulaemus subgenus are proposed based on the largest molecular data set ever used for this clade, which includes 188 individuals and 14 loci representing different parts of the genome (mtDNA, anonymous nuclear loci and nuclear protein‐coding loci). This data set was analysed using two species tree approaches (*beast and MDC). Levels of discordance among methods were found, and with previously published studies, but results are robust enough to propose new phylogenetic hypotheses for the Eulaemus clade. Specifically well‐resolved and well‐supported novel hypotheses are provided within the lineomaculatus section, and we formally recognize the zullyae clade, the sarmientoi clade and the hatcheri group. We also resolve species relationships within the montanus section, and particularly within the melanops series. We found discordance between mitochondrial and nuclear trees and discussed alternative hypotheses for the lineomaculatus and montanus sections, as well as the challenge in resolving phylogenetic relationships for large clades in general.

Molecular phylogeny of the New World gecko genus Homonota (Squamata: Phyllodactylidae)

The genus Homonota was described by Gray (1845) and currently includes 10 species: Homonota andicola, H. borellii, H. darwinii, H. fasciata, H. rupicola, H. taragui, H. underwoodi, H. uruguayensis, H. williamsii & H. whitii and one subspecies of H. darwinii (H. darwinii macrocephala). It is distributed from 15° latitude south in southern Brazil, through much of Bolivia, Paraguay, Uruguay and Argentina to 54° south in Patagonia and across multiple different habitats. Several morphological taxonomic studies on a subset of these species have been published, but no molecular phylogenetic hypotheses are available for the genus. The objective of this study is to present a molecular phylogenetic hypothesis for all the described species in the genus. We sequenced two mitochondrial genes (cyt‐b & 12S: 1745 bp), seven nuclear protein coding (RBMX, DMLX, NKTR, PLRL, SINCAIP, MXRA5, ACA4: 5804 bp) and two anonymous nuclear loci (30Hb, 19Hb: 1306 bp) and implemented traditional concatenated analyses (MP, ML, BI) as well as species‐tree (*beast) approaches. All methods recovered almost the same topology. We recovered the genus Homonota as monophyletic with strong statistical support. Within Homonota, there are three strongly supported clades (whitii, borellii and fasciata), which differ from those previously proposed based on scale shape, osteology, myology and quantitative characters. Detailed morphological analyses based on this highly resolved and well‐supported phylogeny will provide a framework for understanding morphological evolution and historical biogeography of this phenotypically conservative genus. We hypothesize that extensive marine transgressions during Middle and Late Miocene most probably isolated the ancestors of the three main clades in eastern Uruguay (borellii group), north‐western Argentina‐southern Bolivia (fasciata group), and central‐western Argentina (whitii group). Phylogeographic and morphological/morphometric analyses coupled with paleo‐niche modelling are needed to better understand its biogeographical history.

Species tree estimation of North American chorus frogs (Hylidae: Pseudacris) with parallel tagged amplicon sequencing

The field of phylogenetics is changing rapidly with the application of high-throughput sequencing to non-model organisms. Cost-effective use of this technology for phylogenetic studies, which often include a relatively small portion of the genome but several taxa, requires strategies for genome partitioning and sequencing multiple individuals in parallel. In this study we estimated a multilocus phylogeny for the North American chorus frog genus Pseudacris using anonymous nuclear loci that were recently developed using a reduced representation library approach. We sequenced 27 nuclear loci and three mitochondrial loci for 44 individuals on 1/3 of an Illumina MiSeq run, obtaining 96.5% of the targeted amplicons at less than 20% of the cost of traditional Sanger sequencing. We found heterogeneity among gene trees, although four major clades (Trilling Frog, Fat Frog, crucifer, and West Coast) were consistently supported, and we resolved the relationships among these clades for the first time with strong support. We also found discordance between the mitochondrial and nuclear datasets that we attribute to mitochondrial introgression and a possible selective sweep. Bayesian concordance analysis in BUCKy and species tree analysis in *BEAST produced largely similar topologies, although we identify taxa that require additional investigation in order to clarify taxonomic and geographic range boundaries. Overall, we demonstrate the utility of a reduced representation library approach for marker development and parallel tagged sequencing on an Illumina MiSeq for phylogenetic studies of non-model organisms.

Development of anonymous nuclear markers from Illumina paired-end data for Seychelles caecilian amphibians (Gymnophiona: Indotyphlidae)

Abstract Anonymous nuclear markers were developed for Seychelles caecilian amphibians. Using a previously published bioinformatics pipeline (developed for Roche 454 data), 36 candidate anonymous nuclear loci (ANL) of at least 180 bp length were identified from Illumina MiSeq next generation sequencing data for five Seychelles species. We designed primer pairs for the 36 candidate ANL and tested these by PCR and Sanger sequencing. Seven ANL amplified and sequenced well for at least five of the six nominal Seychelles caecilian species (in three genera), and represent potentially useful markers for systematics and conservation.

Random non-coding fragments of lizard DNA: anonymous nuclear loci for the Australian skink, Tiliqua rugosa, and their utility in other Egernia-group species

We report the development of 48 anonymous nuclear loci from the Australian skink Tiliqua rugosa using 454 sequencing. These loci amplified across a Western Australian lineage (47 loci), a ‘northern’ lineage (48 loci) and a ‘southern’ lineage (46 loci). We further tested amplification for the related T. adelaidensis and Egernia stokesii where 37 and 34 loci amplified respectively. The loci showed variability within T. rugosa (22 polymorphic loci) and at least 27 loci also exhibited variation among the three species, highlighting the usefulness of these markers for phylogenetic, phylogeographic and population genetic analyses in T. rugosa and related species.

Intercontinental genetic structure in the amphi-Pacific peatmossSphagnum miyabeanum(Bryophyta: Sphagnaceae)

Unlike seed plants where global biogeographical patterns typically involve interspecific phylogenetic history, spore-producing bryophyte species often have intercontinental distributions that are best understood from a population genetic perspective. We sought to understand how reproductive processes, especially dispersal, have contributed to the intercontinental ‘Pacific Rim’ distribution of Sphagnum miyabeanum. In total, 295 gametophyte plants from western North America (California, Oregon, British Columbia, Alaska), Russia, Japan, and China were genotyped at 12 microsatellite loci. Nucleotide sequences were obtained for seven anonymous nuclear loci plus two plastid regions from 21 plants of S. miyabeanum and two outgroup species. We detected weak but significant genetic differentiation among plants from China, Japan, Alaska, British Columbia, and the western USA. Alaskan plants are genetically most similar to Asian plants, and British Columbian plants are most similar to those in the western USA. There is detectable migration between regions, with especially high levels between Alaska and Asia (China and Japan). Migration appears to be recent and/or ongoing, and more or less equivalent in both directions. There is weak (but significant) isolation-by-distance within geographical regions, and the slope of the regression of genetic on geographical distance differs for Asian versus North American plants. A distinctive Vancouver Island morphotype is very weakly differentiated, and does not appear to be reproductively isolated from plants of the normal morphotype. The intercontinental geographical range of S. miyabeanum reflects recent and probably ongoing migration, facilitated by the production of tiny spores capable of effective long distance dispersal. The results of the present study are consistent with Pleistocene survival of S. miyabeanum in unglaciated Beringia, although we cannot eliminate the possibility that the species recolonized Alaska from Asia more recently.

Deep genetic divergence between disjunct Refugia in the Arctic-Alpine King's Crown, Rhodiola integrifolia (Crassulaceae).

Despite the strength of climatic variability at high latitudes and upper elevations, we still do not fully understand how plants in North America that are distributed between Arctic and alpine areas responded to the environmental changes of the Quaternary. To address this question, we set out to resolve the evolutionary history of the King’s Crown, Rhodiola integrifolia using multi-locus population genetic and phylogenetic analyses in combination with ecological niche modeling. Our population genetic analyses of multiple anonymous nuclear loci revealed two major clades within R. integrifolia that diverged from each other ~ 700 kya: one occurring in Beringia to the north (including members of subspecies leedyi and part of subspecies integrifolia), and the other restricted to the Southern Rocky Mountain refugium in the south (including individuals of subspecies neomexicana and part of subspecies integrifolia). Ecological niche models corroborate our hypothesized locations of refugial areas inferred from our phylogeographic analyses and revealed some environmental differences between the regions inhabited by its two subclades. Our study underscores the role of geographic isolation in promoting genetic divergence and the evolution of endemic subspecies in R. integrifolia. Furthermore, our phylogenetic analyses of the plastid spacer region trnL-F demonstrate that among the native North American species, R. integrifolia and R. rhodantha are more closely related to one another than either is to R. rosea. An understanding of these historic processes lies at the heart of making informed management decisions regarding this and other Arctic-alpine species of concern in this increasingly threatened biome.

Hidden diversity in the Andes: Comparison of species delimitation methods in montane marsupials

Cryptic genetic diversity is a significant challenge for systematists faced with ever-increasing amounts of DNA sequence data. Computationally intensive coalescent-based analyses involving multiple unlinked loci are the only currently viable methods by which to assess the extent to which phenotypically similar populations (or metapopulations) are genetically distinct lineages. Although coalescent-based approaches have been tested extensively via simulations, few empirical studies have examined the impact of prior assumptions and dataset size on the ability to assess genetic isolation (evolutionary independence) using molecular data alone. Here, we consider the efficacy of two coalescent-based approaches (BPP and SpeDeSTEM) for testing the evolutionary independence of cryptic mtDNA haplogroups within three morphologically diagnosable species of Andean mouse opossums (Thylamys pallidior, T. sponsorius, and T. venustus). Fourteen anonymous nuclear loci, one X-linked nuclear intron, and one mitochondrial gene were analyzed for multiple individuals within each haplogroup of interest. We inferred individual gene trees for each locus and considered all of the nuclear loci jointly in a species-tree analysis. Using only the nuclear loci, we performed “species validation” tests for the cryptic mitochondrial lineages in SpeDeSTEM and BPP. For BPP, we also tested a wide range of prior assumptions, assessed performance of the rjMCMC algorithm, and examined how many loci were necessary to confidently delimit lineages. Results from BPP provided strong support for two independent evolutionary lineages each within T. pallidior, T. sponsorius, and T. venustus, whereas SpeDeSTEM results did not support splitting out mtDNA haplogroups as distinct evolutionary units. For most tests, BPP was robust to prior assumptions, although priors were shown to have an effect on both the strength of lineage recognition among T. venustus haplotypes and on the efficiency of the rjMCMC algorithm. Comparisons of results from datasets with different numbers of loci revealed that some cryptic lineages could be confidently delimited with as few as two loci.