Genetic data on endangered twaite shad (Clupeidae) assessed in landlocked and anadromous populations: one or more species?

Seven Italian populations of twaite shad Alosa fallax from Northern and Central Italy were investigated to assess genetic diversity by Cytochrome b (Cytb) gene sequencing. The two ecotypes historically referred to landlocked and anadromous populations were investigated for the first time from a genetic point of view, to clarify their phylogenetic relationships. Moreover, results obtained from populations coming from separated Adriatic and Tyrrhenian basins were compared with data assessed in samples of allis shad Alosa alosa from the Atlantic basin. All the Italian samples were recognized at species level as A. fallax, differing for five mutations from A. alosa. The analyses confirmed the occurrence of a single phylogenetic lineage and of a single species within Italian waters, in both landlocked and migratory populations. The minimum spanning network identified six haplotypes for A. fallax and two haplotypes for A. alosa. The neighbour-joining tree and the maximum likelihood on the Cytb gene sequences confirmed two distinct lineages for A. alosa and A. fallax, without evidence of a separation at specific level within the A. fallax group. A weak separation due to incipient population differentiation was detected between anadromous and landlocked Italian populations, supporting the idea of a recent separation. The molecular data herein collected do not support the existence of the already controversial incipient species Alosa agone. Despite this, the two ecotypes could be considered as different management units from a conservation viewpoint.     

Isolation and characterization of eight dinucleotide microsatellite loci from two closely related clupeid species (Alosa alosa and A. fallax)

Eight dinucleotide microsatellite loci were developed through an enrichment protocol for allis shad (Alosa alosa) and twaite shad (A. fallax). Cross‐species amplification was successful for all loci isolated. The number of alleles per locus ranged from three to nine for A. alosa and from two to seven for A. fallax, while the observed heterozygosity ranged from 0.267 to 0.926 and from 0.240 to 0.727, respectively. These markers will constitute useful tools for studies of population structure and gene flow between two closely related hybridizing species.     

Tolerance of young allis shad Alosa alosa (Clupeidae) to oxy-thermic stress

The ecology of the young stages of allis shad Alosa alosa is poorly documented, although they can be exposed to many pressures during their freshwater phase and their downstream migration. When passing through systems such as the Gironde-Garonne-Dordogne watershed (GGD, SW France), they can be subjected to high temperatures and low levels of oxygen (hypoxia). The aim of this work is to assess the tolerance of young Alosa alosa at four ages (c. 10, 30, 60 and 85 days old) by challenging them to different temperatures (18, 22, 26 and 28°C) together with decreasing oxygen saturation levels (from 100% to 30%). Survival of the 10-day-old individuals was not influenced by oxy-thermic conditions, but high stress levels were detected and perhaps this age class was too fragile regarding the constraint of the experimental design. Survival at 30 and at 60 days old was negatively influenced by the highest temperatures tested alone (from 26°C and from 28°C, respectively) but no effect was detected at 85 days old up to 28°C. A combined effect of temperature and oxygen level was highlighted, with heat accelerating survival decrease when associated with oxygen level depletion: essentially, survival was critical (<50%) at 30 days old at temperature ≥22°C together with 30% O₂; at 60 days old, at temperature = 28°C with 30% O₂; at 85 days old, at temperature ≥26°C with ≤40% O₂. Tolerance to oxy-thermic pressures appeared to be greater among the migratory ages (60 and 85 days old) than among the 30-day-old group. Based on environmental data recorded in the GGD system and on our experimental results, an exploratory analysis allowed a discussion of the possible impact of past oxy-thermic conditions on the local population dynamics between 2005 and 2018. The oxy-thermic conditions that may affect Alosa alosa at ages when they migrate downstream (60 and 85 days old) were not frequently recorded in this period, except in cases of extreme episodes of heat together with hypoxia that occurred in some years, in summertime in the turbidity maximum zone of the Gironde estuary (particularly in the year 2006). Interestingly, oxy-thermic conditions that are likely to threaten the 30-day-old individuals occurred more frequently in the lower freshwater parts of the GGD system between the years 2005 and 2018. In the context of climate change, a general increase in temperature is predicted, as well as more frequent and severe hypoxic events, therefore we suggest that local Alosa alosa population recruitment could encounter critical oxy-thermic conditions more frequently in the future if no adaptive management of water resources occurs.     

Interspecific differentiation and intraspecific substructure in two closely related clupeids with extensive hybridization, Alosa alosa and Alosa fallax

In this study, the evolutionary relationships within and among populations of European shads, Alosa alosa and Alosa fallax , was investigated. Screening of allelic variation across eight allozyme loci and sequencing 448 bp of the mtDNA cytochrome b gene in 14 rivers throughout the range of the species supported that the two taxa were independent lineages (1·3% net nucleotide divergence) despite extensive hybridization. Genetic diversity and structure was considerably higher in A. fallax than A. alosa and the former species revealed evidence of distinct lineages in the Mediterranean and Atlantic basins. A Bayesian clustering approach combined with gill raker counts verified that individuals of the two species could be assigned to their parent group with relatively high confidence. Evaluation of hybridization in the Lima and Mondego Rivers in Portugal provided evidence that introgression is extensive but is not currently obscuring (through hybrid swarming) the diagnosability of the two species.     

Spawning behaviour of Allis shad Alosa alosa: new insights based on imaging sonar data

Spawning behaviour of Alosa alosa was observed by high resolution imaging sonar. Detected clouds of sexual products and micro bubbles served as a potential indicator of spawning activity. Peak spawning time was between 0130 and 0200 hours at night. Increasing detections over three consecutive nights were consistent with sounds of mating events (bulls) assessed in hearing surveys in parallel to the hydro acoustic detection. In 70% of the analysed mating events there were no additional A. alosa joining the event whilst 70% of the mating events showed one or two A. alosa leaving the cloud. In 31% of the analysed mating events, however, three or more A. alosa were leaving the clouds, indicating that matings are not restricted to a pair. Imaging sonar is suitable for monitoring spawning activity and behaviour of anadromous clupeids in their spawning habitats.     

Human disturbance causes the formation of a hybrid swarm between two naturally sympatric fish species

Most evidence for hybrid swarm formation stemming from anthropogenic habitat disturbance comes from the breakdown of reproductive isolation between incipient species, or introgression between allopatric species following secondary contact. Human impacts on hybridization between divergent species that naturally occur in sympatry have received considerably less attention. Theory predicts that reinforcement should act to preserve reproductive isolation under such circumstances, potentially making reproductive barriers resistant to human habitat alteration. Using 15 microsatellites, we examined hybridization between sympatric populations of alewife (Alosa pseudoharengus) and blueback herring (A. aestivalis) to test whether the frequency of hybridization and pattern of introgression have been impacted by the construction of a dam that isolated formerly anadromous populations of both species in a landlocked freshwater reservoir. The frequency of hybridization and pattern of introgression differed markedly between anadromous and landlocked populations. The rangewide frequency of hybridization among anadromous populations was generally 0–8%, whereas all landlocked individuals were hybrids. Although neutral introgression was observed among anadromous hybrids, directional introgression leading to increased prevalence of alewife genotypes was detected among landlocked hybrids. We demonstrate that habitat alteration can lead to hybrid swarm formation between divergent species that naturally occur sympatrically, and provide empirical evidence that reinforcement does not always sustain reproductive isolation under such circumstances.     

Genetic diversity and structure of two hybridizing anadromous fishes (Alosa pseudoharengus,Alosa aestivalis) across the northern portion of their ranges

Alewife (Alosa pseudoharengus) and blueback herring (Alosa aestivalis), also known collectively as either river herring or gaspereau, are anadromous clupeid fishes that display spatiotemporal overlap during riverine spawning migrations. Both species have experienced severe population declines within portions of their ranges. Evidence that they home to their natal rivers to spawn suggests the likelihood of ecologically significant population structure, yet this hypothesis has not been rigorously tested. We examined genetic diversity, differentiation and population structure in 34 alewife and four blueback herring populations spanning a 2,500 km portion of their northern range, using 14 microsatellite loci. Significant differentiation was detected among most rivers, and eight genetically defined alewife population clusters that largely corresponded to hydrographic regions were identified. Similar population structure was seen for blueback herring. Genetic isolation by distance was not significant among alewife populations in regions that have been historically influenced by dams, and/or stock transfers, but was highly significant in two regions that have not been subject to these influences. Genetic differentiation of alewife populations was strongest in the Bay of Fundy. Bottleneck tests revealed evidence of demographic bottlenecks in all of the alewife populations. Lastly, our results indicated that hybridization between alewife and blueback herring is common.     

Electrophoretic identity between allis shad, Alosa alosa (L.), and twaite shad, A.fallax (Lacépède)

Morphological characteristics as well as electrophoretic polymorphism have been analysed in eight samples of allis shad, Alosa alosa (L.) and twaite shad, Alosa fallax (Lacepede), collected in the Loire basin and the Gironde-Dordogne system. The morphological characteristics showed that allis and twaite shad were present in these samples. Moreover, specimens with intermediate characteristics were found in the Loire and assumed to be hybrids between the two forms. By contrast, the two species were monomorphic and electrophoretically indistingishable at the 22 loci analysed. Therefore, it cannot be excluded that these two forms correspond to a single species.     

ELECTROPHORETIC CONFIRMATION OF INTERSPECIFIC HYBRIDIZATION IN AESCULUS (HIPPOCASTANACEAE) AND THE GENETIC STRUCTURE OF A BROAD HYBRID ZONE

Within a broad (>200 km wide) hybrid zone involving three parapatric species of Aesculus, we observed coincident clines in allele frequency for 6 of 14 electrophoretic loci. The cooccurrence of alleles characteristic of A. pavia, A. sylvatica, and A. flava was used to estimate genetic admixtures in 48 populations involving various hybrids between these taxa in the southeastern United States. High levels of allelic polymorphism (up to 40% greater than the parental taxa) were observed in hybrid populations and also in some populations bordering the hybrid zone. A detailed analysis of a portion of the hybrid zone involving A. pavia and A. sylvatica revealed a highly asymmetrical pattern of gene flow, predominantly from Coastal Plain populations of A. pavia into Piedmont populations of A. sylvatica. Computer simulations were used to generate expected genotypic arrays for parental, F₁; and backcross individuals, which were compared with natural populations using a character index scoring system. In these comparisons, hybrid individuals could be distinguished from either parent, but F₁ and backcross progeny could not be distinguished from each other. Most hybrid populations were found to include hybrids and one of the parental taxa, but never both parents. Three populations appeared to be predominantly hybrids with no identifiable parental individuals. Hybrids occurred commonly at least 150 km beyond the range of A. pavia, but usually not more than 25 km beyond the range of A. sylvatica. Introgression, suggested by genetically hybrid individuals and significant gene admixtures of two or more species in populations lacking morphological evidence of hybridization, may extend the hybrid zone further in both directions. The absence of one or both parental species from hybrid populations implies a selective disadvantage to parentals in the hybrid zone and/or that hybridization has occurred through long-distance gene flow via pollen, primarily from A. pavia into A. sylvatica. Long-distance pollen movement in plants may generate hybrid zones of qualitatively different structure than those observed in animals, where gene flow involves dispersal of individuals.     

Maintenance of strong morphological differentiation despite ongoing natural hybridization between sympatric species of Lomatia (Proteaceae)

Background and Aims

When species cohesion is maintained despite ongoing natural hybridization, many questions are raised about the evolutionary processes operating in the species complex. This study examined the extensive natural hybridization between the Australian native shrubs Lomatia myricoides and L. silaifolia (Proteaceae). These species exhibit striking differences in morphology and ecological preferences, exceeding those found in most studies of hybridization to date.

Methods

Nuclear microsatellite markers (nSSRs), genotyping methods and morphometric analyses were used to uncover patterns of hybridization and the role of gene flow in morphological differentiation between sympatric species.

Key Results

The complexity of hybridization patterns differed markedly between sites, however, signals of introgression were present at all sites. One site provided evidence of a large hybrid swarm and the likely presence of multiple hybrid generations and backcrosses, another site a handful of early generational hybrids and a third site only traces of admixture from a past hybridization event. The presence of cryptic hybrids and a pattern of morphological bimodality amongst hybrids often disguised the extent of underlying genetic admixture.

Conclusions

Distinct parental habitats and phenotypes are expected to form barriers that contribute to the rapid reversion of hybrid populations to their parental character state, due to limited opportunities for hybrid/intermediate advantage. Furthermore, strong genomic filters may facilitate continued gene flow between species without the danger of assimilation. Stochastic fire events facilitate temporal phenological isolation between species and may partly explain the bi-directional and site-specific patterns of hybridization observed. Furthermore, the findings suggest that F₁ hybrids are rare, and backcrosses may occur rapidly following these initial hybridization events.     

Genetic analysis of admixture and patterns of introgression in foundation cottonwood trees (Salicaceae) in southwestern Colorado,USA

Cottonwoods are well known as foundation riparian trees that support diverse communities and drive ecosystem processes. Although hybridization naturally occurs when the distributions of two or more cottonwood species overlap, few cottonwood hybrid zones have been genetically characterized. We use genetic and genomic analyses to characterize patterns of admixture and introgression for a newly described hybrid zone at the intersection of three species (Populus L. Salicaceae—Populus deltoides, Populus fremontii, and Populus angustifolia) in southwestern Colorado, USA. Analysis of nuclear and chloroplast microsatellite marker data detected substantial genetic variation among individuals, revealing that (1) hybridization is occurring between two, not three, species (P. deltoides and P. angustifolia); (2) gene flow is bidirectional; (3) hybrids are not abundant (admixture detected in only 34 of 270 trees), with most being early-generation F₁ hybrids; (4) cytonuclear disequilibria exists and F₁ hybrids tend to retain P. deltoides—like chloroplasts; and (5) roughly 30 % of the nuclear markers deviated from a neutral pattern of introgression, suggesting that selection may play a role in shaping the genetic structure of the hybrid zone in this region. Overall, our results show that despite strong selection maintaining species divergence, transfer of allelic variants across species boundaries can occur. Our study assesses the fine-scale genetic structure of hybridization between P. angustifolia and P. deltoides and lays the foundation for examining how geographic differences in hybrid zone dynamics arise and may influence subsequent ecological and evolutionary processes.     

Migration behaviour of twaite shad Alosa fallax assessed by otolith Sr:Ca and Ba:Ca profiles

Individual migration behaviour during the juvenile and adult life phase of the anadromous twaite shad Alosa fallax in the Elbe estuary was examined using otolith Sr:Ca and Ba:Ca profiles. Between hatching and the end of the first year of life, juveniles showed two migration patterns. Pattern one exhibited a single downstream migration from fresh water to the sea with no return into fresh water. In contrast, pattern two showed a first migration into the sea, then a return into fresh water and, finally, a second downstream migration into marine water. This first report of migration plasticity for A. fallax points to different exposure times to estuarine threats depending on the migration strategy. In adults, high Sr:Ca and low Ba:Ca in the majority of individuals confirmed prior reports of a primarily marine habitat use. Patterns reflecting spawning migrations were rarely observed on otoliths, possibly due to the short duration of visits to fresh water.     

Microsatellite DNA markers for American shad (Alosa sapidissima) and cross‐species amplification within the family Clupeidae

Sixteen microsatellite loci were identified and characterized for American shad (Alosa sapidissima). The number of alleles per locus observed ranged from eight to 32 and averaged 15.4 alleles. Average observed heterozygosity was 81.1%. The markers were screened using four other species from the family Clupeidae. Amplification success among Alosa species was 79.2% with 81.6% polymorphism among those markers that amplified successfully. Amplification success was poor in Dorosoma (31.3%). Due to allelic diversity and estimates of heterozygosity, these markers can be useful in A. sapidissima for population level analyses, parentage assignment and broodstock management.     

First evidence of natural reproduction of the Allis shad Alosa alosa in the River Rhine following re‐introduction measures

After stocking with larvae from the Gironde–Garonne–Dordogne population, in 2013, three young‐of‐the‐year Allis shad Alosa alosa, probably originating from natural reproduction, were documented for the first time in a period of nearly 100 years in the River Rhine. In 2014, a further increase was observed when 57 juveniles and eight adults were caught; seven of these eight adults were derived from stocking, indicating the success of stocking measures within the framework of the EU‐LIFE project.     

Multigenerational outbreeding effects in Chinook salmon (Oncorhynchus tshawytscha)

Outbreeding, mating between genetically divergent individuals, may result in negative fitness consequences for offspring via outbreeding depression. Outbreeding effects are of notable concern in salmonid research as outbreeding can have major implications for salmon aquaculture and conservation management. We therefore quantified outbreeding effects in two generations (F₁ hybrids and F₂ backcrossed hybrids) of Chinook salmon (Oncorhynchus tshawytscha) derived from captively-reared purebred lines that had been selectively bred for differential performance based on disease resistance and growth rate. Parental lines were crossed in 2009 to create purebred and reciprocal hybrid crosses (n = 53 families), and in 2010 parental and hybrid crosses were crossed to create purebred and backcrossed hybrid crosses (n = 66 families). Although we found significant genetic divergence between the parental lines (F_ST = 0.130), reciprocal F₁ hybrids showed no evidence of outbreeding depression (hybrid breakdown) or favorable heterosis for weight, length, condition or survival. The F₂ backcrossed hybrids showed no outbreeding depression for a suite of fitness related traits measured from egg to sexually mature adult life stages. Our study contributes to the current knowledge of outbreeding effects in salmonids and supports the need for more research to better comprehend the mechanisms driving outbreeding depression.     

Genetic polymorphism of adenosine deaminase (ADA; E.C. 3.5.4.4.) in allis shad, Alosa alosa and twaite shad, Alosa fallax

A genetic polymorphism of adenosine deaminase (ADA), is described for the first time in European shad species, Alosa alosa and Alosa fallax . Significant heterogeneity in gene frequencies was found, both between species and within species.     

The presence of anadromous twaite shad (Alosa fallax) in the Ebro River (western Mediterranean,Spain): an indicator of the population's recovery?

Although the Ebro River is one of the largest Mediterranean rivers, the presence of anadromous fish species was near to complete extirpation since the second half of the 20th century. However, many mature twaite shads (Alosa fallax) were captured at the lower reaches of this river (Xerta's weir site, 63 river km) in spring 2005, suggesting a natural recovery of this population. In this study, the first biological data on twaite shad from the Ebro River are presented, and their significance for the species conservation and recovery are discussed. The historical reasons for the decline of this species are reviewed, and data supports the proposal for urgent construction of a proper fish passage for anadromous species at the Xerta's weir. This action would allow fish access to a further 60 km of river upstream of the weir, representing a 200% increase in the amount of freshwater habitat and potential spawning areas. Improved access together with restrictive fishery regulations will assist with renewed interest in the fishery and conservation of the species.     

Asymmetric introgression between Magnolia stellata and M. salicifolia at a site where the two species grow sympatrically

In order to understand the ongoing evolutionary relationships between species, it is important to elucidate patterns of natural hybridization. In the zone where two species are sympatrically distributed, we examined 274 individuals of Magnolia stellata, Magnolia salicifolia, and their putative hybrids by means of 16 nuclear and three chloroplast microsatellite markers. Hybrid classes of individuals were estimated by admixture analyses. Morphological traits were also investigated for 64 of the 274 individuals. Admixture analyses revealed that 66 of the 274 individuals were classified as hybrids, comprising 17 F₁ and 19 F₂ individuals, 27 backcrosses to M. salicifolia, and 3 individuals of unknown origin. Morphological data from the 64 individuals agreed well with their genetic admixture rates. Spatial locations of F₁ and F₂ hybrids at the study site were intermediate between the two purebred species, indicating that the site preferences of hybrids are intermediate. The occurrences of F₂ and backcross hybrids indicate that F₁ hybrids are fertile. The chloroplast DNA haplotypes of all F₁ hybrids corresponded to those detected in M. salicifolia, so that maternal parents of the F₁ hybrids were all M. salicifolia. Furthermore, no hybrid individuals derived from a backcross to M. stellata were detected. These results suggest that the direction of hybridization and the subsequent introgression have been quite asymmetric and that the introgression occurred from M. stellata into M. salicifolia.     

Boundaries and hybridization in a secondary contact zone between freshwater mussel species (Family:Unionidae)

Correct species identification and delineation are crucial for effective conservation and management. However, species delineation can be problematic in the presence of morphological ambiguities due to phenotypic plasticity, convergence, and/or interspecific hybridization. Here, we investigated the degree of hybridization between two closely related freshwater mussel species [Bivalvia: Unionidae; Lampsilis siliquoidea (Barnes) and L. radiata (Gmelin)] that present intermediate forms in areas of sympatry. Unionids have a distinct form of mitochondrial DNA (mtDNA) inheritance, termed doubly uniparental inheritance (DUI) where female mtDNA (F-type) is transmitted to all progeny but male mtDNA (M-type) is mostly inherited by the males resulting in mostly homoplasmic females and heteroplasmic males. An individual was identified as hybrid when F-type and M-type mtDNA of the two different species were found in the same individual. Twelve out of 116 sequenced males were identified as hybrids indicating that these species hybridize where their geographic range overlaps in the lower Great Lakes and St. Lawrence basins. Microsatellite analyses further support the occurrence of hybridization but at a larger spatial scale than indicated by the mitochondrial analyses. We also found that strong within-species population genetic structure affects the detection of purebred individuals overestimating the number of hybrids. Given the large geographic scale and proportion of hybrids found in this study, natural hybridization and introgression need to be considered when implementing local biodiversity inventories, identifying waterbodies as source of organisms for relocation and restoration projects and when setting appropriate conservation policies.Subject terms: Evolutionary genetics, Genetic hybridization     

Transgressive character expression in hybrid zones between the native invasives Tithonia tubaeformis and Tithonia rotundifolia (Asteraceae) in Mexico

Natural hybridization frequently promotes gene introgression among closely related species in sympatric populations, producing complex patterns of morphological variation. Therefore, a detailed understanding of the dynamics of interspecific gene flow and its morphological patterns is of widespread interest. We tested if introgressive hybridization promotes an increase in transgressive characters in comparison with the parental species. A sunflower species complex occurring in Mexico formed by two native invasive species, Tithonia tubaeformis and Tithonia rotundifolia, was analyzed using 46 morphological characters (leaf, flower and fruit) in five hybrid zones (N = 150 individuals) and two pure sites for each parental species (N = 80 individuals). In general, T. tubaeformis differed significantly from T. rotundifolia in all the examined characters, except six foliar and one inflorescence character. Morphological characters support the hypothesis of hybridization in this complex, even though both species remain morphological distinct in mixed stands. Individual hybrids appear to be a mosaic of parent-like (24.8 % of traits), intermediate (26.1 %) and transgressive (37.8 %) phenotypes (the remaining 11.3 % of the traits did not differ significantly from both parental species). Our results suggest that individuals from the same parental species were more similar among themselves than to putative hybrids, indicating occasional hybridization with segregation in hybrid types or backcrossing to parents. Evidence indicates a unidirectional pattern of gene flow toward T. rotundifolia.