Species delimitation with gene flow: A methodological comparison and population genomics approach to elucidate cryptic species boundaries in Malaysian Torrent Frogs

Accurately delimiting species boundaries is a nontrivial undertaking that can have significant effects on downstream inferences. We compared the efficacy of commonly used species delimitation methods (SDMs) and a population genomics approach based on genomewide single‐nucleotide polymorphisms (SNPs) to assess lineage separation in the Malaysian Torrent Frog Complex currently recognized as a single species (Amolops larutensis). First, we used morphological, mitochondrial DNA and genomewide SNPs to identify putative species boundaries by implementing noncoalescent and coalescent‐based SDMs (mPTP, iBPP, BFD*). We then tested the validity of putative boundaries by estimating spatiotemporal gene flow (fastsimcoal2 , ABBA‐BABA) to assess the extent of genetic isolation among putative species. Our results show that the A. larutensis complex runs the gamut of the speciation continuum from highly divergent, genetically isolated lineages (mean F_st = 0.9) to differentiating populations involving recent gene flow (mean F_st = 0.05; N_m > 5). As expected, SDMs were effective at delimiting divergent lineages in the absence of gene flow but overestimated species in the presence of marked population structure and gene flow. However, using a population genomics approach and the concept of species as separately evolving metapopulation lineages as the only necessary property of a species, we were able to objectively elucidate cryptic species boundaries in the presence of past and present gene flow. This study does not discount the utility of SDMs but highlights the danger of violating model assumptions and the importance of carefully considering methods that appropriately fit the diversification history of a particular system.     

Separation in flowering time contributes to the maintenance of sympatric cryptic plant lineages

Sympatric cryptic lineages are a challenge for the understanding of species coexistence and lineage diversification as well as for management, conservation, and utilization of plant genetic resources. In higher plants studies providing insights into the mechanisms creating and maintaining sympatric cryptic lineages are rare. Here, using microsatellites and chloroplast sequence data, morphometric analyses, and phenological observations, we ask whether sympatrically coexisting lineages in the common wetland plant Juncus effusus are ecologically differentiated and reproductively isolated. Our results show two genetically highly differentiated, homoploid lineages within J. effusus that are morphologically cryptic and have similar preference for soil moisture content. However, flowering time differed significantly between the lineages contributing to reproductive isolation and the maintenance of these lineages. Furthermore, the later flowering lineage suffered less from predispersal seed predation by a Coleophora moth species. Still, we detected viable and reproducing hybrids between both lineages and the earlier flowering lineage and J. conglomeratus, a coexisting close relative. Flowering time differentiation between the lineages can be explained by neutral divergence alone and together with a lack of postzygotic isolation mechanisms; the sympatric coexistence of these lineages is most likely the result of an allopatric origin with secondary contact.     

Contrasting abundance and contribution of clonal proliferation to the population structure of the corkscrew sea anemone Bartholomea annulata in the tropical Western Atlantic

Clonal propagation is an important life history trait for many sessile organisms, and often leads to the formation of monoclonal aggregations. In the marine environment, sea anemones have been model species for testing theory regarding the evolution of sex and understanding the contribution of sexual versus asexual reproduction to the population structure in facultatively clonal animals. However, little attention has been paid to tropical actiniarians. The corkscrew anemone Bartholomea annulata is common in tropical marine habitats in the western Atlantic and Caribbean; it forms small aggregations (2–4 anemones) on coral reefs and larger aggregations (>10 anemones) in mangrove habitats. We used field surveys and molecular analyses to investigate patterns of distribution, abundance, and genetic structure of aggregations formed by B. annulata on a reef in the US Virgin Islands and in a unique mangrove habitat in the Florida Keys. Abundance was greatest at the abandoned rock quarry mangrove habitat in the Florida Keys, where anemones formed continuously distributed aggregations carpeting the exposed limestone walls. Genetic diversity assessed via intersimple sequence repeats (ISSRs) and six microsatellite loci revealed that asexual reproduction plays only a minor role in the formation of both small and large anemone aggregations. Specifically, ISSR analyses showed that only ~10% of anemone aggregations were clonal in the US Virgin Islands, while microsatellite genotyping identified clonality in only 1 of 35 aggregations. In the Florida Keys, only four clonal genotypes were recovered within aggregations, but eight clones, representing 33% of the total surveyed population, had individuals in multiple aggregations. Thus, population structure of B. annulata appears to rely primarily on sexual reproduction, although asexual reproduction may play a nontrivial role in some environments. Mechanisms that drive the formation of genotypically diverse aggregations remain unknown, but may include attraction toward conspecifics, shared use of preferred habitats, or the local retention of larvae in partially enclosed habitats.     

Out of sight, out of mind: high cryptic diversity obscures the identities and histories of geminate species in the marine bivalve subgenus Acar

Aim The rise of the Isthmus of Panama and the formation of ‘geminate’ species pairs serves as an important model of allopatric speciation. However, to function as a model system, hypothesized geminates must first be shown to be each other’s closest living relatives. If the presence of cryptic taxa obscures true relationships, the biogeographical histories of transisthmian taxa are likely to be misinterpreted. We have therefore completed a phylogeographic survey of the transisthmian bivalve subgenus Acar in the genus Barbatia to characterize patterns of tropical American diversity and to place transisthmian taxa in a regional phylogeographic context. Location Tropical America. Methods Mitochondrial cytochrome c oxidase I (COI) and nuclear internal transcribed spacer (ITS) sequences were obtained from 233 specimens of Acar. Sequences were analysed using cladistic and Bayesian methods. Divergence times between species were inferred from net nucleotide divergences and a coalescence‐based method. Results The survey revealed 22 COI clades that were also monophyletic at ITS, indicating that the taxonomy of Acar is potentially greater than a fivefold diversity underestimate. The lone previously recognized geminate [Barbatia (Acar) gradata and Barbatia (Acar) domingensis] is composed of 15 clades. Among the four transisthmian lineages identified, two diverged more than 14 Ma; the two other geminates split just prior to the time of final seaway closure. In addition to a fourfold increase in the number of known geminates, our data show that within‐basin diversification has been more impressive, with one geminate splitting into five monophyletic clades in the Western Atlantic alone since seaway closure. Electron microscopy of the larval shells of specimens indicates that the transisthmian lineage with the greatest rate of post‐Isthmian diversification possesses non‐planktonic larvae, a life‐history feature linked to high speciation rates. Main conclusions Our analyses revealed that the identities of geminate pairs split by the Isthmus of Panama were obscured by extremely high tropical American cryptic diversity. Although we have identified four geminates, only two appear to have been split by the Isthmus. Our uncovering of extensive post‐Isthmian diversification is consistent with the palaeontological perspective that the final closure of the Central American Seaway was followed by high rates of subgeneric diversification, particularly in the tropical Western Atlantic.     

Patterns and processes in the evolutionary history of parrotfishes (Family Labridae)

Phylogenetic reconstruction of the evolutionary relationships among 61 of the 70 species of the parrotfish genera Chlorurus and Scarus (Family Labridae) based on mitochondrial and nuclear gene sequences retrieved 15 well‐supported clades with mid Pliocene/Pleistocene diversification. Twenty‐two reciprocally monophyletic sister‐species pairs were identified: 64% were allopatric, and the remainder were sympatric. Age of divergence was similar for allopatric and sympatric species pairs. Sympatric sister pairs displayed greater divergence in morphology, ecology, and sexually dimorphic colour patterns than did allopatric pairs, suggesting that both genetic drift in allopatric species pairs and ecologically adaptive divergence between members of sympatric pairs have played a role in diversification. Basal species typically have small geographical ranges and are restricted to geographically and ecologically peripheral reef habitats. We found little evidence that a single dominant process has driven diversification, nor did we detect a pattern of discrete, sequential stages of diversification in relation to habitat, ecology, and reproductive biology. The evolution of Chlorurus and Scarus has been complex, involving a number of speciation processes. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 107 , 529–557.     

Cryptic diversity and comparative phylogeography of the estuarine copepod Acartia tonsa on the US Atlantic coast

Unexpectedly strong geographic structures in many cosmopolitan species of marine holoplankton challenge the traditional view of their unrestrained dispersal and presumably high gene flow. We investigated cryptic lineage diversity and comparative phylogeography of a common estuarine copepod, Acartia tonsa, on the US Atlantic coast, using mitochondrial (mtCOI) and nuclear (nITS) gene markers. Three broadly sympatric lineages (F, S, X) were defined by genealogically concordant clades across both gene trees, strongly supporting recognition as reproductively isolated species. Limited dispersal seems to have had a major role in population differentiation of A. tonsa in general, with gene flow propensities rank ordered X > S > F. Geographic structure was found only at large scales (1000–2000 km) in X and S. Phylogeographic patterns in all three lineages were mostly concordant with previously recognized zoogeographic provinces but a large mid‐Atlantic gap in the occurrence of lineage X, coupled with its presence in Europe, suggests possible nonindigenous origins. For lineage F, physiological adaptation to low‐salinity environments is likely to have accentuated barriers to gene flow and allopatric differentiation at both regional and continental scales. Three allopatric F sublineages inferred a southern centre of origin and a stepwise northward diversification history at the continental scale. The most recently derived F sublineages, in the mid‐Atlantic Bight, showed strong phylogeographic patterns at nITS albeit weaker at mtCOI. Applying a crustacean mtCOI molecular clock suggests that A. tonsa lineages diverged pre‐Pleistocene but mid‐Atlantic F lineage diversification may be post‐Pleistocene.     

Are sympatrically speciating Midas cichlid fish special? Patterns of morphological and genetic variation in the closely related species Archocentrus centrarchus

Established empirical cases of sympatric speciation are scarce, although there is an increasing consensus that sympatric speciation might be more common than previously thought. Midas cichlid fish are one of the few substantiated cases of sympatric speciation, and they formed repeated radiations in crater lakes. In contrast, in the same environment, such radiation patterns have not been observed in other species of cichlids and other families of fish. We analyze morphological and genetic variation in a cichlid species (Archocentrus centrarchus) that co‐inhabits several crater lakes with the Midas species complex. In particular, we analyze variation in body and pharyngeal jaw shape (two ecologically important traits in sympatrically divergent Midas cichlids) and relate that to genetic variation in mitochondrial control region and microsatellites. Using these four datasets, we analyze variation between and within two Nicaraguan lakes: a crater lake where multiple Midas cichlids have been described and a lake where the source population lives. We do not observe any within‐lake clustering consistent across morphological traits and genetic markers, suggesting the absence of sympatric divergence in A. centrarchus. Genetic differentiation between lakes was low and morphological divergence absent. Such morphological similarity between lakes is found not only in average morphology, but also when analyzing covariation between traits and degree of morphospace occupation. A combined analysis of the mitochondrial control region in A. centrarchus and Midas cichlids suggests that a difference between lineages in the timing of crater lake colonization cannot be invoked as an explanation for the difference in their levels of diversification. In light of our results, A. centrarchus represents the ideal candidate to study the genomic differences between these two lineages that might explain why some lineages are more likely to speciate and diverge in sympatry than others.     

Temporally consistent species differences in parasite infection but no evidence for rapid parasite‐mediated speciation in Lake Victoria cichlid fish

Parasites may have strong eco‐evolutionary interactions with their hosts. Consequently, they may contribute to host diversification. The radiation of cichlid fish in Lake Victoria provides a good model to study the role of parasites in the early stages of speciation. We investigated patterns of macroparasite infection in a community of 17 sympatric cichlids from a recent radiation and 2 older species from 2 nonradiating lineages, to explore the opportunity for parasite‐mediated speciation. Host species had different parasite infection profiles, which were only partially explained by ecological factors (diet, water depth). This may indicate that differences in infection are not simply the result of differences in exposure, but that hosts evolved species‐specific resistance, consistent with parasite‐mediated divergent selection. Infection was similar between sampling years, indicating that the direction of parasite‐mediated selection is stable through time. We morphologically identified 6 Cichlidogyrus species, a gill parasite that is considered a good candidate for driving parasite‐mediated speciation, because it is host species‐specific and has radiated elsewhere in Africa. Species composition of Cichlidogyrus infection was similar among the most closely related host species (members of the Lake Victoria radiation), but two more distantly related species (belonging to nonradiating sister lineages) showed distinct infection profiles. This is inconsistent with a role for Cichlidogyrus in the early stages of divergence. To conclude, we find significant interspecific variation in parasite infection profiles, which is temporally consistent. We found no evidence that Cichlidogyrus‐mediated selection contributes to the early stages of speciation. Instead, our findings indicate that species differences in infection accumulate after speciation.     

Early evolutionary history of the flowering plant family Annonaceae: steady diversification and boreotropical geodispersal

Aim Rain forest‐restricted plant families show disjunct distributions between the three major tropical regions: South America, Africa and Asia. Explaining these disjunctions has become an important challenge in biogeography. The pantropical plant family Annonaceae is used to test hypotheses that might explain diversification and distribution patterns in tropical biota: the museum hypothesis (low extinction leading to steady accumulation of species); and dispersal between Africa and Asia via Indian rafting versus boreotropical geodispersal. Location Tropics and boreotropics. Methods Molecular age estimates were calculated using a Bayesian approach based on 83% generic sampling representing all major lineages within the family, seven chloroplast markers and two fossil calibrations. An analysis of diversification was carried out, which included lineage‐through‐time (LTT) plots and the calculation of diversification rates for genera and major clades. Ancestral areas were reconstructed using a maximum likelihood approach that implements the dispersal–extinction–cladogenesis model. Results The LTT plots indicated a constant overall rate of diversification with low extinction rates for the family during the first 80 Ma of its existence. The highest diversification rates were inferred for several young genera such as Desmopsis, Uvariopsis and Unonopsis. A boreotropical migration route was supported over Indian rafting as the best fitting hypothesis to explain present‐day distribution patterns within the family. Main conclusions Early diversification within Annonaceae fits the hypothesis of a museum model of tropical diversification, with an overall steady increase in lineages possibly due to low extinction rates. The present‐day distribution of species within the two largest clades of Annonaceae is the result of two contrasting biogeographic histories. The ‘long‐branch clade’ has been diversifying since the beginning of the Cenozoic and underwent numerous geodispersals via the boreotropics and several more recent long‐distance dispersal events. In contrast, the ‘short‐branch clade’ dispersed once into Asia via the boreotropics during the Early Miocene and further dispersal was limited.     

Divergent mitochondrial lineages arose within a large,panmictic population of the Savannah sparrow (Passerculus sandwichensis)

Unusual patterns of mtDNA diversity can reveal interesting aspects of a species’ biology. However, making such inferences requires discerning among the many alternative scenarios that could underlie any given mtDNA pattern. Next‐generation sequencing methods provide large, multilocus data sets with increased power to resolve unusual mtDNA patterns. A mtDNA‐based phylogeography of the Savannah sparrow (Passerculus sandwichensis) previously identified two sympatric, but divergent (~2%) clades within the nominate subspecies group and a third clade that consisted of birds sampled from northwest Mexico. We revisited the phylogeography of this species using a population genomic data set to resolve the processes leading to the evolution of sympatric and divergent mtDNA lineages. We identified two genetic clusters in the genomic data set corresponding to (a) the nominate subspecies group and (b) northwestern Mexico birds. Following divergence, the nominate clade maintained a large, stable population, indicating that divergent mitochondrial lineages arose within a panmictic population. Simulations based on parameter estimates from this model further confirmed that this demographic history could produce observed levels of mtDNA diversity. Patterns of divergent, sympatric mtDNA lineages are frequently interpreted as admixture of historically isolated lineages. Our analyses reject this interpretation for Savannah sparrows and underscore the need for genomic data sets to resolve the evolutionary mechanisms behind anomalous, locus‐specific patterns.     

Nuclear DNA recapitulates the cryptic mitochondrial lineages of Lumbricus rubellus and suggests the existence of cryptic species in an ecotoxological soil sentinel

Mitochondrial DNA analysis has revealed two distinct phylogenetic lineages within the ecotoxological sentinel earthworm model Lumbricus rubellus Hoffmeister, 1843. The existence of these lineages could complicate ecotoxicological studies that use the species as a sentinel for soil contamination testing, as they may respond differently to contamination; however, as mitochondrial haplotypes are not always expected to segregate in the same way as chromosomal DNA in natural populations, we further investigated this issue by using nuclear DNA markers (microsatellites) to measure genetic diversity, differentiation, and gene flow in sympatric populations of the two L. rubellus lineages at two sites in South Wales. Our results show that sympatric populations of the two lineages are more genetically differentiated than geographically distant populations of the same lineage, and Bayesian clustering analysis revealed no evidence of gene flow between the lineages at either site. Additionally, DNA sequencing of these microsatellite loci uncovered substantial differentiation between lineages at homologous flanking regions. Overall our findings indicate a high degree of nuclear genetic differentiation between the two lineages of L. rubellus, implying reproductive isolation at the two study sites and therefore the potential existence of cryptic species. The existence of two cryptic taxa has major implications for the application of L. rubellus as an ecotoxicological sentinel. It may therefore be necessary to consider the lineages as separate taxa during future ecotoxicological studies. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 110 , 780–795.     

Temperate and tropical algal-sea anemone symbioses

Abstract. In this review, we seek to develop new insights about the nature of algal‐sea anemone symbioses by comparing such associations in temperate and tropical seas. Temperate seas undergo pronounced seasonal cycles in irradiance, temperature, and nutrients, while high irradiance, high temperature, and low nutrients are seasonally far less variable in tropical seas. We compare the nature of symbiosis between sea anemones (= actinians) and zooxanthellae (Symbiodinium spp.) in both regions to test tropical paradigms against temperate examples and to identify directions for future research. Although fewer anemone species are symbiotic in temperate regions, they are locally dominant and ecologically important members of the benthic community compared to the tropics. Zooxanthella densities tend to be lower in temperate anemones, but data are limited to a few species in both temperate and tropical seas. Zooxanthella densities are far more stable over time in temperate anemones than in tropical anemones, suggesting that temperate symbioses are more resistant to fluctuations in environmental parameters such as irradiance and temperature. Light‐saturated photosynthetic rates of temperate and tropical zooxanthellae are similar, but temperate anemone hosts receive severely reduced carbon supplies from zooxanthellae during winter months when light is reduced. Symbiont transmission modes and specificity do not show any trends among anemones in tropical vs. temperate seas. Our review indicates the need for the following: (1) Investigations of other temperate and tropical symbiotic anemone species to assess the generality of trends seen in a few “model’ anemones. (2) Attention to the field ecology of temperate and tropical algal‐anemone symbioses, for example, how symbioses function under seasonally variable environmental factors and how zooxanthellae persist at high densities in darkness and winter. The greater stability of zooxanthella populations in temperate hosts may be useful to understanding tropical symbioses in which bleaching (loss of zooxanthellae) is of major concern. (3) Study of the evolutionary history of symbiosis in both temperate and tropical seas. Continued exploration of the phylogenetic relationships between host anemones and zooxanthella strains may show how and why zooxanthellae differ in anemone hosts in both environments.     

The comparative biogeography of Philippine geckos challenges predictions from a paradigm of climate‐driven vicariant diversification across an island archipelago

A primary goal of biogeography is to understand how large‐scale environmental processes, like climate change, affect diversification. One often‐invoked but seldom tested process is the “species‐pump” model, in which repeated bouts of cospeciation are driven by oscillating climate‐induced habitat connectivity cycles. For example, over the past three million years, the landscape of the Philippine Islands has repeatedly coalesced and fragmented due to sea‐level changes associated with glacial cycles. This repeated climate‐driven vicariance has been proposed as a model of speciation across evolutionary lineages codistributed throughout the islands. This model predicts speciation times that are temporally clustered around the times when interglacial rises in sea level fragmented the islands. To test this prediction, we collected comparative genomic data from 16 pairs of insular gecko populations. We analyze these data in a full‐likelihood, Bayesian model‐choice framework to test for shared divergence times among the pairs. Our results provide support against the species‐pump model prediction in favor of an alternative interpretation, namely that each pair of gecko populations diverged independently. These results suggest the repeated bouts of climate‐driven landscape fragmentation have not been an important mechanism of speciation for gekkonid lizards across the Philippine Archipelago.     

Patterns of diversification in two species of short‐tailed bats (Carollia Gray, 1838): the effects of historical fragmentation of Brazilian rainforests

The small‐sized frugivorous bat Carollia perspicillata is an understory specialist and occurs in a wide range of lowland habitats, tending to be more common in tropical dry or moist forests of South and Central America. Its sister species, Carollia brevicauda, occurs almost exclusively in the Amazon rainforest. A recent phylogeographic study proposed a hypothesis of origin and subsequent diversification for C. perspicillata along the Atlantic coastal forest of Brazil. Additionally, it also found two allopatric clades for C. brevicauda separated by the Amazon Basin. We used cytochrome b gene sequences and a more extensive sampling to test hypotheses related to the origin and diversification of C. perspicillata plus C. brevicauda clade in South America. The results obtained indicate that there are two sympatric evolutionary lineages within each species. In C. perspicillata, one lineage is limited to the Southern Atlantic Forest, whereas the other is widely distributed. Coalescent analysis points to a simultaneous origin for C. perspicillata and C. brevicauda, although no place for the diversification of each species can be firmly suggested. The phylogeographic pattern shown by C. perspicillata is also congruent with the Pleistocene refugia hypothesis as a likely vicariant phenomenon shaping the present distribution of its intraspecific lineages. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 102 , 527–539.     

Phylogenetic analysis,taxonomic revision,and dental ontogeny of the Cretaceous Zhelestidae (Mammalia: Eutheria)

Iphisa elegans Gray, 1851 is a ground‐dwelling lizard widespread over Amazonia that displays a broadly conserved external morphology over its range. This wide geographical distribution and conservation of body form contrasts with the expected poor dispersal ability of the species, the tumultuous past of Amazonia, and the previously documented prevalence of cryptic species in widespread terrestrial organisms in this region. Here we investigate this homogeneity by examining hemipenial morphology and conducting phylogenetic analyses of mitochondrial (CYTB) and nuclear (C‐MOS) DNA sequence data from 49 individuals sampled across Amazonia. We detected remarkable variation in hemipenial morphology within this species, with multiple cases of sympatric occurrence of distinct hemipenial morphotypes. Phylogenetic analyses revealed highly divergent lineages corroborating the patterns suggested by the hemipenial morphotypes, including co‐occurrence of different lineages. The degrees of genetic and morphological distinctness, as well as instances of sympatry among mtDNA lineages/morphotypes without nuDNA allele sharing, suggest that I. elegans is a complex of cryptic species. An extensive and integrative taxonomic revision of the I. elegans complex throughout its wide geographical range is needed. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 166 , 361–376.     

Embracing discordance: Phylogenomic analyses provide evidence for allopolyploidy leading to cryptic diversity in a Mediterranean Campanula (Campanulaceae) clade

The Mediterranean Basin harbors a remarkable amount of biodiversity, a high proportion of which is endemic to this region. Here, we present an in‐depth study of an angiosperm species complex, in which cryptic taxonomic diversity has been hypothesized. Specifically, we focus on four currently recognized species in the Roucela complex, a well‐supported clade in the Campanulaceae/Campanuloideae: Campanula creutzburgii, C. drabifolia, C. erinus, and C. simulans. This study takes a phylogenomic approach, utilizing near‐complete plastomes and 130 nuclear loci, to uncover cryptic diversity and test hypotheses regarding hybridization and polyploidy within this clade. Genome size estimates recovered tetraploid and octoploid lineages within the currently recognized, widespread species C. erinus, showing an east‐west geographic pattern. Though genomic data clearly differentiate these two cytotypes, we failed to discern morphological differences. The formation of a cryptic octoploid lineage, distributed across the eastern Mediterranean, is hypothesized to be the result of an allopolyploid event in which one parental morphology is retained. The tetraploid C. erinus and C. creutzburgii (also a tetraploid) are implicated as parental lineages. Our results highlight the utility of target‐enrichment approaches for obtaining genomic datasets for thorough assessments of species diversity and the importance of carefully considering gene‐tree discordance within such datasets.     

Speciation in ninespine stickleback: reproductive isolation and phenotypic divergence among cryptic species of Japanese ninespine stickleback

Although similar patterns of phenotypic diversification are often observed in phylogenetically independent lineages, differences in the magnitude and direction of phenotypic divergence have been also observed among independent lineages, even when exposed to the same ecological gradients. The stickleback family is a good model with which to explore the ecological and genetic basis of parallel and nonparallel patterns of phenotypic evolution, because there are a variety of populations and species that are locally adapted to divergent environments. Although the patterns of phenotypic divergence as well as the genetic and ecological mechanisms have been well characterized in threespine sticklebacks, Gasterosteus aculeatus, we know little about the patterns of phenotypic diversification in other stickleback lineages. In eastern Hokkaido, Japan, there are three species of ninespine sticklebacks, Pungitius tymensis and the freshwater type and the brackish‐water type of the P. pungitius–P. sinensis species complex. They utilize divergent habitats along coast–stream gradients of rivers. Here, we investigated genetic, ecological and phenotypic divergence among three species of Japanese ninespine sticklebacks. Divergence in trophic morphology and salinity tolerance occurred in the direction predicted by the patterns observed in threespine sticklebacks. However, the patterns of divergence in armour plate were different from those previously found in threespine sticklebacks. Furthermore, the genetic basis of plate variation may differ from that in threespine sticklebacks. Because threespine sticklebacks are well‐established model for evolutionary research, the sympatric trio of ninespine sticklebacks will be an invaluable resource for ecological and genetic studies on both common and lineage‐specific patterns of phenotypic diversification.     

Reticulate evolution and sea‐level fluctuations together drove species diversification of slipper orchids (Paphiopedilum) in South‐East Asia

South‐East Asia covers four of the world's biodiversity hotspots, showing high species diversity and endemism. Owing to the successive expansion and contraction of distribution and the fragmentation by geographical barriers, the tropical flora greatly diversified in this region during the Tertiary, but the evolutionary tempo and mode of species diversity remain poorly investigated. Paphiopedilum, the largest genus of slipper orchids comprising nearly 100 species, is mainly distributed in South‐East Asia, providing an ideal system for exploring how plant species diversity was shaped in this region. Here, we investigated the evolutionary history of this genus with eight cpDNA regions and four low‐copy nuclear genes. Discordance between gene trees and network analysis indicates that reticulate evolution occurred in the genus. Ancestral area reconstruction suggests that vicariance and long‐distance dispersal together led to its current distribution. Diversification rate variation was detected and strongly correlated with the species diversity in subg. Paphiopedilum (~80 species). The shift of speciation rate in subg. Paphiopedilum was coincident with sea‐level fluctuations in the late Cenozoic, which could have provided ecological opportunities for speciation and created bridges or barriers for gene flow. Moreover, some other factors (e.g. sympatric distribution, incomplete reproductive barriers and clonal propagation) might also be advantageous for the formation and reproduction of hybrid species. In conclusion, our study suggests that the interplay of reticulate evolution and sea‐level fluctuations has promoted the diversification of the genus Paphiopedilum and sheds light into the evolution of Orchidaceae and the historical processes of plant species diversification in South‐East Asia.