Species Boundaries within Morphologically Cryptic Galagos: Evidence from Acoustic and Genetic Data

<p>Understanding the evolutionary forces that shape populations in the marine environment is critical for predicting population dynamics and dispersal patterns for marine organisms. For organisms with complex reproductive strategies, this remains a challenge. Sponges fulfil many functional roles and are important components of benthic environments in tropical, temperate and polar oceans. They have evolved diverse reproductive strategies, reproducing both sexually and asexually, and thus provide an opportunity to investigate complicated evolutionary questions. This PhD thesis examines sexual and asexual reproduction in two common golf-ball sponges in central New Zealand (Tethya bergquistae and T. burtoni), with particular focus on how the environment influences these modes of reproduction, and further, how they shape species delineations and connectivity patterns. New Zealand waters are projected to experience increases in temperature and decreases in nutrients over the next century, and therefore these species may be experience changes in basic organismal processes like reproduction due to climate change, requiring adaptation to local environments. Therefore, this work has important implications when considering how reproductive phenology, genetic diversity and population structure of marine populations may change with shifts in climate. In my first data chapter, I highlight the difficulty in delineating sponge species by investigating the evolutionary relationship of Tethya spp. in central New Zealand using both morphological and molecular methods. Phylogenetic reconstructions based on two mitochondrial markers (rnl, COI-ext) and one nuclear marker (18S) revealed three genetic clades, with one clade representing T. bergquistae and two clades belonging to what was a priori thought to be a single species, T. burtoni. Morphological analysis based on spicule characteristics allowed T. bergquistae to be distinguished from T. burtoni, but revealed no apparent differences between the T. burtoni clades. These results indicate hidden genetic diversity within T. burtoni, which likely represents a group consisting of incipient species that have undergone speciation but have yet to express clear morphological differences. This chapter supports the notion that cryptic speciation in sponges may go undetected and diversity underestimated when using only morphology-based taxonomy, a result which has implications for conservation and management of marine systems. In my second data chapter, I characterize the reproductive biology for both species of Tethya in relation to potential environmental drivers, including sea surface temperature, chlorophyll-a concentration and rainfall. Using histological methods for sponges collected monthly over two years, Tethya spp. were found to be gonochoristic and oviparous sexual reproducers, with one annual reproductive event occurring in the austral summer from January to March. Differences in oocyte density and reproductive output between both species and sites highlighted both species-specific adaptive responses and environmental influences on reproduction. Temperature and rainfall were found to be correlated with instances of sexual reproduction, and the summer reproductive event occurred each year following the spring bloom of chlorophyll-a. These findings indicate that seasonal fluctuations in the environment may be important for triggering gametogenesis for these species. With shifts in temperature, productivity, and timing of seasons projected for New Zealand, there is a potential for reproductive phenology to become mismatched with the surrounding environment under future climate change scenarios, which has consequences for the frequency, duration and overall output of sexual reproduction for these sponges. My third data chapter characterizes asexual reproduction in both species of Tethya, exploring relationships between reproductive traits and potential environmental drivers that may influence asexual budding events. Two sponge populations, one for each species of Tethya, were monitored over two years by both monthly sampling and periodic in situ observations. Data revealed that budding occurred continuously throughout the year, but had a cyclic pattern where instances of budding and densities of buds were higher during the austral spring and summer. Asexual reproduction coincided with sexual reproduction, and some individuals were found to simultaneously reproduce using both modes. Instances of asexual reproduction were positively associated with temperature and rainfall, but distinct differences between species were difficult to identify. As temperature proved important, an experiment looking at bud production in relation to thermal stress was conducted, where sponges were subjected to stable temperatures treatments of 17°C (control), 19°C and 21°C. No instances of budding were observed under any temperature treatment, and high mortality occurred in the 21°C treatment. These results suggest that temperature changes (i.e., heterogeneous environments) may be more important than temperature alone in driving asexual reproduction, and further, indicate thermal stress will result in increased sponge mortality. Correlations to potential environmental drivers indicate that future shifts in climate may affect instances of asexual reproduction and thus sponge abundance, which has the potential to alter the genetic structure and overall diversity of these populations. In the final data chapter, I developed novel microsatellite markers for Tethya burtoni to characterize the genetic connectivity patterns among four populations in central New Zealand, with particular interest in the roles that sexual and asexual reproduction play in connectivity. I sampled three sites within 10 km of each other in the Wellington Region (WR), and another site on an island (Kapiti Island) approximately 50 km north of the WR. At one of the WR sample sites, I monitored a T. burtoni population over two years to examine the dispersal range of asexually reproduced buds and the ability of clones to sexually reproduce. The WR and Kapiti Island populations were strongly genetically differentiated, but within the WR region, two populations were genetically similar, indicative of high connectivity. For the monitored population, asexual bud dispersal was restricted to no greater than 1 m and clonal individuals had reduced sexual reproductive ability. Asexual reproduction did not appear to play an important role in interpopulation connectivity nor gene flow, as buds had low dispersal ability and rarely reproduced. Population structure and connectivity for T. burtoni appear to be largely driven by sexual reproduction, and asexual reproduction instead aids genotype survivorship and population maintenance. These findings highlight that different reproductive modes can differentially contribute to population dynamics in sessile marine organisms, suggesting that predictions about future population viability under changing environments may be difficult to make. In summary, this PhD thesis uses a combination of genetic, histological, field-based and experimental methods to examine species boundaries, reproduction and connectivity for Tethya spp. on rocky reefs of New Zealand. The sympatric nature, complex reproductive ecology and connectivity patterns observed likely shape the complex evolutionary processes occurring in these sponges, including introgressive hybridization and cryptic species. Individuals that showed evidence of possible introgressive events occurred mainly in populations with more restricted gene flow, while the presence of both cryptic species were more prevalent in well connected populations. Such a trend allows for discussion of under what circumstances both of these processes occur. Furthermore, environmental correlates to both sexual and asexual reproduction indicate that both of these modes of reproduction have the potential to be altered with future changes in the environment. As both modes were found to play different roles in gene flow within and between populations, future shifts in climate are also expected to alter population structure and connectivity for these sponges. Such shifts in gene flow will also likely result in changes to species boundaries and thus the overall diversity of this genus. Many other sessile, benthic marine organisms present reproductive traits and behaviours similar to those of Tethya spp., and therefore these results can aid in the interpretation of results for other marine taxa. Overall, this thesis describes the population dynamics of Tethya spp., which are abundant and ecologically important on New Zealand reefs, and provides insight on how temperate sponge populations may fare with climate change, which has important implications for management and conservation efforts.</p>

Cryptic species – genetically distinct species that are morphologically difficult to distinguish – present challenges to systematists. Operationally, cryptic species are very difficult to identify and sole use of genetic data or morphological data can fail to recognize evolutionarily isolated lineages. We use morphometric data to test species boundaries hypothesized with genetic data in the North Caribbean bark anole (Anolis distichus), a suspected species complex. We use univariate and multivariate analyses to test if candidate species based on genetic data can be accurately diagnosed. We also test alternative species delimitation scenarios with a model fitting approach that evaluates normal mixture models capable of identifying morphological clusters. Our analyses reject the hypothesis that the candidate species are diagnosable. Neither uni- nor multivariate morphometric data distinguish candidate species. The best-supported model included two morphological clusters; however, these clusters were uneven and did not align with a plausible species divergence scenario. After removing two related traits driving this result, only one cluster was supported. Despite substantial differentiation revealed by genetic data, we recover no new evidence to delimit species and refrain from taxonomic revision. This study highlights the importance of considering other types of data along with molecular data when delimiting species.

Recent advances in morphometrics and genetics have led to the discovery of numerous cryptic species in coral reef ecosystems. A prime example is the Montastraea annularis scleractinian coral species complex, in which morphological, genetic, and reproductive data concur on species boundaries, allowing evaluation of long-term patterns of speciation and evolutionary innovation. Here we test for cryptic species in the Atlantic species, Montastraea cavernosa, long recognized as polymorphic. Our modern samples consist of 94 colonies collected at four locations (Belize, Panama, Puerto Rico in the Caribbean; Sao Tome in the Eastern Atlantic). Our fossil samples consist of 78 colonies from the Plio-Pleistocene of Costa Rica and Panama. Landmark morphometric data were collected on thin sections of 46 modern and 78 fossil colonies. Mahalanobis distances between colonies were calculated using Bookstein coordinates, revealing two modern and four fossil morphotypes. The remaining 48 of the 94 modern colonies were assigned to morphotype using discriminant analysis of calical measurements. Cross-tabulation and multiple comparisons tests show no significant morphological differences among geographic locations or water depths. Patterns of variation within and among fossil morphotypes are similar to modern morphotypes. DNA sequence data were collected for two polymorphic nuclear loci (β-tub1 and β-tub2) on all 94 modern colonies. Haplotype networks show that both genes consist of two clades, but morphotypes are not associated with genetic clades. Genotype frequencies and two-locus genotype assignments indicate genetic exchange across clades, and ϕst values show no genetic differentiation between morphotypes at different locations. Taken together, our morphological and genetic results do not provide evidence for cryptic species in M. cavernosa, but indicate instead that this species has an unusually high degree of polymorphism over a wide geographic area and persisting for >25 million years (myr).

Editorial and Retrospective 2008

The aim of this study was to determine the influence of biogeography, oceanography and ecology on the population structure of different crustacean species with emphasis on their larval development. It is predicted that the duration of larval development influences the dispersal capability and therefore the level of genetic differentiation. In addition, the results should provide answers if there are cryptic species. The two atlanto-mediterranean crab species Xantho hydrophilus (Herbst, 1790) and Xantho poressa (Olivi, 1792) and the European prawn Palaemon elegans (Rathke, 1837) were included as marine species. Xantho has a shorter larval development with four zoeal stages than Palaemon with nine larval stages. Therefore, a higher gene flow within Palaemon was expected. We used also the amphidromous species Xiphocaris elongata (Guérin-Méneville, 1856) which occurs in freshwater systems of the Caribbean islands. The life cycle of amphidromous X. elongata is intermediate between freshwater and marine species and thus, high genetic differentiation was predicted. A comparative population genetics analyses was conducted to reveal differences in the population structure due to the distinct larval development and the amphidromous life-cycle. In the case of X. hydrophilus, results show a restricted gene flow between the populations of the Atlantic and the Mediterranean Sea. However, the taxonomic status of the Mediterranean subspecies X. h. granulicarpus is not valid as long as no single mutation step was detected to distinguish constantly between the two subspecies X. hydrophilus and X. h. granulicarpus. In addition, morphological transitional forms of X. hydrophilus are found within the Western Mediterranean Sea. X. poressa is suggested to reproduce panmictically because no differences are found between the populations of the Mediterranean Sea and the Atlantic Ocean and a mark recapture experiment showed that the species reaches high population densities. This species is well adapted to its habitat due to its variability in colouration and size which is the result of passive defence (camouflage). A pattern of allometric growth in the carapace shape was observed. Phylogenetic analyses revealed that X. sexdentatus is very closely related to X. hydrophilus. This could be either due to hybridization or introgression. Despite the fact that Palaemon elegans has nine larval stages, there is surprisingly high genetic differentiation. Normally such strong differences are only found between species and thus a cryptic species within the P. elegans complex is suggested. Three types of haplotype groups are found: Type I from the Atlantic and the Alboran Sea, Type II only within the Mediterranean Sea and Type III within the Mediterranean, Black, Caspian, Baltic and East Sea. Type III could be a relict of the Messinian crisis due to its high genetic differentiation, while Type II has recolonized the Mediterranean Sea after the crisis and ongoing separation mechanisms have established restricted gene flow between Type I and II. The barrier to gene flow between the Atlantic Type and the two Mediterranean Types is the Almería-Oran-Front. The Almería-Oran-Front is an important phylogeographic break, where restricted gene flow is possible. An isolation-by-distance pattern could be detected within the Atlantic Ocean. Furthermore, there is significant genetic differentiation between the Northern Atlantic and the North-East Atlantic due to the English Channel. These results provide evidence that dispersal play a minor role in determining the genetic structure of a species, compared to the biogeographic history and physical factors. This becomes even more obvious by comparison with the shrimp Xiphocaris elongata, for which a higher genetic differentiation due to the isolated freshwater systems and the amphidromous life cycle was expected. Although there is a high genetic differentiation, no geographic pattern emerges. The amphidromous life cycle enhance the dispersal capability and gene flow. It displays rather patterns of a marine species than of an exclusively freshwater species. The species is characterized by its variability in the rostrum length, but the results give no evidence for a genetic differentiation of the phenotypes. Thus, adaptive plasticity due to predators and in part allometric growth could be an explanation for the variability.

The Indo‐Australian archipelago (IAA) supports the world's highest diversity of marine fish, invertebrates and reptiles. Many of the marine fish and invertebrates show congruent phylogeographic patterns, supporting a view that the region's complex geo‐climatic history has played an important role in generating its exceptional biodiversity. Here, we examine population genetic structure of the viviparous sea snake, Hydrophis curtus, to assess how past and present barriers to gene flow in the IAA have contributed to genetic and species diversity in a fully marine reptile. Mitochondrial and anonymous nuclear sequences and ten microsatellite loci were used to identify patterns of historical genetic structure and population expansion, reconstruct dated genealogies and assess levels of recent gene flow. These markers revealed strong concordant geographic structure within H. curtus with a prominent genetic break between populations broadly distributed in the Indian Ocean and the West Pacific. These populations were estimated to have diverged in the late Pliocene or early Pleistocene, and microsatellite admixture analyses suggested limited recent gene flow between them despite the current lack of barriers to dispersal, indicating possible cryptic species. Subsequent divergence in the mid–late Pleistocene was detected within the West Pacific clade among the populations in the Phuket‐Thailand region, South‐East Asia and Australia, and two of these populations also showed genetic signals of recent range expansions. Our results show that climatic fluctuations during the Plio‐Pleistocene generated high levels of cryptic genetic diversity in H. curtus, and add to similar findings for diverse other marine groups in the IAA.

The microcrustacean Daphnia is arguably one of the most studied zooplankton species, having a well understood ecology, life history, and a relatively well studied evolutionary history. Despite this wealth of knowledge, species boundaries within closely related species in this genus often remain elusive and the major evolutionary forces driving the diversity of daphniids remain controversial. This genus contains more than 80 species with multiple cryptic species complexes, with many closely related species able to hybridize. Here, we review speciation research in Daphnia within the framework of current speciation theory. We evaluate the role of geography, ecology, and biology in restricting gene flow and promoting diversification. Of the 253 speciation studies on Daphnia, the majority of studies examine geographic barriers (55%). While evidence shows that geographic barriers play a role in species divergence, ecological barriers are also probably prominent in Daphnia speciation. We assess the contribution of ecological and nonecological reproductive isolating barriers between closely related species of Daphnia and found that none of the reproductive isolating barriers are restricting gene flow completely. Research on reproductive isolating barriers has disproportionally focused on two species complexes, the Daphnia pulex and Daphnia longispina species complexes. Finally, we identify areas of research that remain relatively unexplored and discuss future research directions that build our understanding of speciation in daphniids.

Recent seismic experiments have revealed that a shallow, low velocity zone is a common feature within the uppermost few hundred meters of very young (less than 100 kyr) oceanic crust at ‘fast spreading’ ridges. The shape of this zone suggests that the low velocities are related to the constructional morphology of the extrusive (layer 2A) portion of the crust. With this in mind, we use simple analytical techniques to study the effects of characteristic morphologic elements of the extrusive basalts on their elastic properties. We find that high compliance of the contacts between pillows and pillow fragments can account for all of the observed velocity anomaly. Voids between pillows are, by comparison to their effects on porosity, considerably less important. Radial (cooling) cracks and voids within the pillows themselves will affect the velocities in direct proportion to their effect on the individual pillows. The relatively rapid increase in velocity with age in these very young extrusives can be explained by welding of pillow contacts and stiffening of the individual pillows by infilling of radial fractures, both of which may take place without a large reduction in porosity. If voids between pillows are filled initially by sediments, the cementation of these materials can also cause large increases in velocity without a large addition of mass. Because of the importance of such subtle variations in morphology, there can be no one‐to‐one relationship between seismic velocities and porosities within the extrusive layer of the oceanic crust. Therefore independent measures of crustal porosity must be found (for example, using sea floor gravity surveys).

Accurate species delimitation is critical for biodiversity conservation. Integrative taxonomy has been advocated for a long time, yet tools allowing true integration of genetic and phenotypic data have been developed quite recently and applied to few models, especially in plants. In this study, we investigated species boundaries within a group of twelve Pseudophrys taxa from France by analyzing genetic, morphometric and chemical (i.e., floral scents) data in a Bayesian framework using the program integrated Bayesian Phylogenetics and Phylogeography (iBPP). We found that these twelve taxa were merged into four species when only genetic data were used, while most formally described species were recognized as such when only phenotypic (either morphometric or chemical) data were used. The result of the iBPP analysis performed on both genetic and phenotypic data supports the proposal to merge Ophrys bilunulata and O. marmorata on the one hand, and O. funerea and O. zonata on the other hand. Our results show that phenotypic data are particularly informative in the section Pseudophrys and that their integration in a model-based method significantly improves the accuracy of species delimitation. We are convinced that the integrative taxonomic approach proposed in this study holds great promise to conduct taxonomic revisions in other orchid groups.

The evolutionary dynamics of cryptic species remain poorly understood, and their detection relies primarily on methods that quantify divergence, assuming that gene flow is absent. Here, we examine how gene flow shapes the evolutionary trajectories and species boundaries in Bornean Fanged Frogs, a renowned example of cryptic diversity where a single species has been split into 18 genetically divergent yet morphologically indistinguishable species. We employed target-capture data from over 13,000 loci to assess lineage independence of 14 nominal species distributed across Malaysian Borneo by evaluating both divergence and cohesion using network multispecies coalescent (NMSC) and MSC + migration approaches. Under the Unified Species Concept, only six of the 14 nominal species unambiguously form independently evolving lineages; the remainder represent cohesive metapopulation lineages nested within those six species. While mitochondrial p-distances varied substantially (up to 10%), genome-wide net divergences (Da) were more consistent, ranging from 0.5-2%, placing all the hypothesized "cryptic species" within the empirical gray zone of the speciation continuum. We show that diversification in the gray zone is unpredictable and heavily impacted by gene flow, leading to two key phenomena that confound species delimitation: (1) the artifactual branch effect, where admixed lineages are inferred as long, early-diverging branches, creating an illusion of deep divergence; and (2) the species-definition anomaly zone, where intraspecific pairwise sequence distances exceed interspecific ones. We further demonstrate that divergence in the gray zone varies among metrics and genomic regions, reflecting heterogeneity in evolutionary dynamics across the genome. Different genomic markers also vary considerably in phylogenetic discordance and their ability to retain signatures of gene flow. Loci from anchored hybrid enrichment (AHE) and ultraconserved elements (UCE) produced less phylogenetic discordance and retained signals of older introgression but failed to detect recent migration, making them suitable for phylogenetic reconstruction and inferring ancient introgression, but not ongoing gene flow. Recognizing the central role of gene flow reframes our understanding of cryptic species; rather than being considered as genetically distinct units that failed to evolve morphological differentiation, they are manifestations of continuous diversification in the gray zone. This shift in perspective offers a new and dynamic evolutionary framework for identifying and interpreting cryptic biodiversity across the Tree of Life.

Portillo, Frank, Branch, William R., Tilbury, Colin R., Nagy, Zoltán T., Hughes, Daniel F., Kusamba, Chifundera, Muninga, Wandege M., Aristote, Mwenebatu M., Behangana, Mathias, Greenbaum, Eli (2019): A Cryptic New Species of Polemon (Squamata: Lamprophiidae, Aparallactinae) from the Miombo Woodlands of Central and East Africa. Copeia 107 (1): 22-35, DOI: 10.1643/ch-18-098, URL: http://dx.doi.org/10.1643/ch-18-098

With the recent attention and focus on quantitative methods for species delimitation, an overlooked but equally important issue regards what has actually been delimited. This study investigates the apparent arbitrariness of some taxonomic distinctions, and in particular how species and subspecies are assigned. Specifically, we use a recently developed Bayesian model-based approach to show that in the Hercules beetles (genus Dynastes) there is no statistical difference in the probability that putative taxa represent different species, irrespective of whether they were given species or subspecies designations. By considering multiple data types, as opposed to relying exclusively on genetic data alone, we also show that both previously recognized species and subspecies represent a variety of points along the speciation spectrum (i.e., previously recognized species are not systematically further along the continuum than subspecies). For example, based on evolutionary models of divergence, some taxa are statistically distinguishable on more than one axis of differentiation (e.g., along both phenotypic and genetic dimensions), whereas other taxa can only be delimited statistically from a single data type. Because both phenotypic and genetic data are analyzed in a common Bayesian framework, our study provides a framework for investigating whether disagreements in species boundaries among data types reflect (i) actual discordance with the actual history of lineage splitting, or instead (ii) differences among data types in the amount of time required for differentiation to become apparent among the delimited taxa. We discuss what the answers to these questions imply about what characters are used to delimit species, as well as the diverse processes involved in the origin and maintenance of species boundaries. With this in mind, we then reflect more generally on how quantitative methods for species delimitation are used to assign taxonomic status.

Modern methods for species delimitation provide biologists with the power to detect cryptic diversity in nearly any system. To illustrate the application of such methods, we collected data (21 sequence loci) from a carnivorous plant in southeastern North America and applied several recently developed methods (Gaussian clustering, Structurama, BPP, spedeSTEM). The pale pitcher plant Sarracenia alata inhabits the southeastern USA along the northern coast of the Gulf of Mexico. Sarracenia alata populations are separated by the Mississippi River and Atchafalaya Basin, a known biogeographical barrier in this region, but the cohesiveness of S. alata as currently classified has not been tested rigorously. Multiple analytical approaches (including allelic clustering and species trees methods) suggest that S. alata comprises two cryptic lineages that correspond to the eastern and western portions of the plant's distribution. That such clear genetic evidence for cryptic diversity exists within S. alata and is in conflict with other sources of data (e.g. morphology, environmental differentiation) illustrates a conundrum faced by those who investigate species boundaries: genetic data are often the first type of data to accumulate evidence of differentiation, but most existing taxonomic treatments are based on nongenetic data. Our results suggest that S. alata as currently described contains two cryptic species, and we recommend the elevation of the western populations to species status. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109, 737–746.

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Species boundaries in scleractinian corals remain highly elusive due to conflicting patterns between morphological and molecular phylogenies, often caused by morphological plasticity, occurrence of cryptic species, incomplete lineage sorting or introgressive hybridization. Here, we use an integrated systematics approach, which combines reduced representation genome sequencing (nextRAD), micro-morphometric characterization, SEM analyses and compilation of life history traits, to infer phylogenetic relationships among closely related species in the Caribbean coral genus Madracis. In total, we analyzed 235 Madracis specimens from Curaçao and Bermuda collected from 10–90 m depth. Sequence- and SNP-based analyses for 115 samples generated unprecedented species resolution in Madracis, greatly supporting the morphology-based taxonomy of the current, accepted Caribbean species M. senaria, M. decactis, M. formosa, M. carmabi and M. mirabilis (M. auretenra). The exception was M. pharensis, in which we found evidence for three separate lineages, and for which we found signatures of admixture and introgression. These three M. pharensis lineages showed distinct depth distributions (thus classified as shallow, deep and very deep) and were partially distinguishable on the basis of fine microstructural elements of the collumella, septa and coenosteum. Further taxonomic comparisons are needed to formalize these putative cryptic species. Overall, our integrated systematics approach further resolves species relationships in the Caribbean genus Madracis, supports the morphological descriptions for most of the recognized species, but also reveals the existence of cryptic diversity in groups marked by high admixture, thus suggesting hybridization as a driving force in coral species diversity.