Zoosyst. Evol. 96 (2) 2020, 797-805 | DOI 10.3897/zse.96.55896 > PENSUFT. thes BERLIN Amphipods in estuaries: the sibling species low salinity switch hypothesis David J. Wildish', Adriana E. Radulovici? 1 Fisheries & Oceans Canada, Biological Station, 531 Brandy Cove Road, St. Andrews, New Brunswick, Canada 2 Centre for Biodiversity Genomics, University of Guelph, Guelph, Ontario, Canada http://zoobank.org/90AF CD24-4D5A-4F74-A9F 4-E8696852C028 Corresponding author: David J. Wildish (talitridnb@gmail.com) Academic editor: Pavel Stoev # Received 26 June 2020 Accepted 12 August 2020 Published 19 November 2020 Abstract A novel low salinity switch hypothesis is proposed to account for the speciation of an obligate estuarine (oligohaline) amphipod, Or- chestia aestuarensis, from a closely-related one, Orchestia mediterranea, found in both estuarine and marine conditions (euryhaline). The underlying genetic mechanisms could involve: 1. Adimorphic allele, or linked set of alleles, carried by the euryhaline amphipod which controls the ability to breed in low salinity conditions in estuaries and which is selected for in these conditions, producing the oligohaline amphipod. 2. A genetically-assimilated gene or genes, controlling the ability to breed in low salinity conditions in estuaries, which is/are “switched on” by low salinity conditions. 3. Allopatric speciation from a euryhaline to an oligohaline amphipod species where low salinity conditions is the selective switch. It is possible that other estuarine, sibling, amphipod pairs have evolved by salinity switching. In the North Atlantic coastal region, this could include: Gammarus tigrinus/G. daiberi and G. salinus/G. zaddachi (Amphipoda, Gammaridae). Key Words Low salinity switch hypothesis, sibling species amphipods, evolution, estuaries, O. mediterranea, O. aestuarensis Introduction The Amphipoda are one of 16 orders of the Crustacea (Horton et al., 2016). Over nine thousand (9,980) species of amphipods have been described to 2016 (Arfianti et al. 2018) occurring in a wide range of habitats: marine, estuarine, freshwater, terrestrial and in special association with other animals, such as parasites. The Amphipoda contain species which extend from the marine environment into estuaries and freshwater. In colo- nising estuaries, the immigrant species demonstrate varying degrees of physiological acclimatisation or adaptation to the reduced salinities, which is the defining feature of estuaries. We review the literature pertinent to sibling species pairs in estuaries and propose the low salinity switch hy- pothesis to explain their evolutionary origin. Our review focuses on the euryhaline talitrid: Orchestia mediterra- nea A. Costa, 1853 (Crustacea, Amphipoda, Talitridae) and its oligohaline sibling, Orchestia aestuarensis, Wild- ish, 1987. A description of the kinds of estuaries that am- phipods might encounter as they colonise them is given. Study methods are reviewed which can examine the in- ference that the oligohaline amphipod evolved by salinity switching from its euryhaline sibling. Types of Estuaries An estuary is that part of the hydrological system where freshwater and marine waters mix. Classification of estu- Copyright David J. Wildish, Adriana E. Radulovici. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 798 aries is based either on how they geologically form or how and where, the waters mix within them (Odum 1971). The classification based on how and where fresh and saltwater mix within the estuary is of most use in this study. Odum (1971) recognised salt wedge, partially mixed and well mixed estuaries. Specialised salinity con- ditions were present in fjords, consisting of a deep basin of very saline, sometimes stagnant seawater. Hypersaline estuaries in the tropics are formed when evaporation ex- ceeds the freshwater inflow. There have been attempts to classify well mixed estuaries by the salinity conditions within the estuary, coupled with the benthic fauna found throughout its length. Thus, the Venice System (Den Har- tog 1960; 1963a) is based on salinity in relation to benthic macro-invertebrate distribution. The Venice System is ap- plicable to coastal plain, well-mixed (lowland) estuaries of the North Atlantic coast of the Old World. The biologi- cally-based (fish and macro-invertebrates) salinity zones, derived by Bulger et al. (1993), apply to partially mixed estuaries of the North Atlantic coast in the New World. Due to the varied physical and hydrological conditions in estuaries, a universal classification, based on salinity distribution, applicable to the varied geological types of estuaries, 1s unlikely to be achieved. In this study, we have relied on direct measurement of chlorinity or salinity in the estuary, accounting for tidal effects to correlate with amphipod distribution. Up until the 1980s, salinity was measured by titrating the halide content with silver nitrate, yielding a “chlorinity” as parts per thousand (ppt), which, if multiplied by 1.80655, gives salinity as ppt. Assuming that typical, undiluted seawater has a salinity of 35g/kg or 35 ppt, the earlier chlorinity measurements can be converted to a percentage of 35 ppt salinity seawater and this is the most useful way of ex- pressing it if salinity tolerance experiments are contem- plated. From the 1980s onwards, salinity measurements were increasingly made by electrical conductivity meth- ods and the results expressed as practical salinity units (PSU). Such measurements may be converted from the relationship: 35 PSU = 100% seawater. Low salinity switch hypothesis The hypothesis, proposed here, is that low salinity acts as an environmental switch by “choosing” the new phe- notype, which can live and reproduce at lower salinities, from amongst adjacent euryhaline, sibling populations which are adapted to live and reproduce in a higher range of salinities. Thus, the new phenotype 1s “induced” by the appropriate salinity conditions in a new estuary from a eu- ryhaline, sibling population reaching the appropriate sa- linity conditions. Specific mechanisms which could con- trol the appearance of an oligohaline from a euryhaline amphipod in low salinity locations in estuaries include: 1. By a polymorphic allele or linked set of alleles, carried at low frequency within a population of the zse.pensoft.net Wildish, D.J. & Radulovici, A.E.: Amphipods in estuaries euryhaline amphipod which is selected for in low salinity conditions. The alleles control an ability to survive and breed in low salinity conditions. This is the combined switch mechanism of West-Eberhard (1989), with an allelic and low salinity switch. 2. After initial polyphenism by the ancestral euryhaline amphipod, the ability to breed at low salinities became fixed by genetic assimilation (Whitman and Agrawal 2000). The same gene or genes are then switched on by the appropriate low salinity conditions. 3. By regular allopatric or parapatric speciation mech- anisms which occur within estuaries. This involves mutation and/or random genetic drift amongst the parental euryhaline amphipod population and selec- tion in low salinity conditions of a more oligohaline amphipod in the appropriate salinity conditions. Kolding (1985) reports on two guilds of Gammarus species in the Baltic Sea which have evolved by al- lopatric speciation in this way over a period of ~ 4000 years. The null hypothesis for each of the hypothetical mecha- nisms above would be that genetic change and natural selec- tion were not involved in the phenotypic changes observed. Criteria for selection of the estuarine sibling salinity switch pair: O. mediterranea/O. aestuarensis Orchestia mediterranea A. Costa, 1857 and O. aestuaren- sis Wildish, 1987 were selected, based on the following biological/geological criteria: ¢ both sibling species occur in the same estuary, but only one is found in the brackish part (oligohaline) and the other in a wider range of estuarine and fully marine habitats (euryhaline). ¢ based on morphological characters, it is difficult to distinguish the siblings taxonomically. ¢ in field conditions, the two siblings are reproduc- tively isolated. ¢ each sibling tolerates a different range of salinities in estuaries. * genetically, the siblings are closer to each other than to other congeneric species and they may be able to hybridise. ¢ because the oligohaline sibling may be of more recent origin, recent geological history may be of importance in determining which physical factors, such as salinity changes, were the ultimate cause of sibling speciation. In what follows, we review, in the same order as listed above, the literature available for each of the six crite- ria as it pertains to the evolution of O. aestuarensis and O. mediterranea in estuaries. Zoosyst. Evol. 96 (2) 2020, 797-805 Geographical distribution A synonomy list for O. mediterranea is given in Wildish (1969) from which the distribution data (Fig. 1) is taken. Summarising, it is a marine, eulittoral species found on north-eastern Atlantic coastal beaches (absent on north-west- er Atlantic beaches) with a northern limit on the German coast at Busum and Hallig Hooge (Schellenberg 1942). It is present on the British, Irish and continental European coasts and, thus, on suitable coastal beaches in the North Sea, Irish Sea, Bay of Biscay, Mediterranean and Black Seas. It 1s ab- sent in the Baltic Sea. The most southerly records are from coastal Tunisia and Algeria in the Mediterranean Sea and from Kossier in the Red Sea (Ruffo 1938a, b). There are reports of O. mediterranea from the Canary and Azores ar- chipelagoes (Dahl 1967). It is also present in estuaries and detailed distribution data within the Deltaic estuaries of 60°N 20°N 20°W 0° 799 Holland are given by Den Hartog (1963b) and in the Med- way Estuary, Britain, by Wildish (1970a). O. aestuarensis was originally described as a morph of O. mediterranea and not given specific status until 1987 (Wildish 1987). To date, it has been identified in the following estuaries: Tamar, Medway (Wildish 1969), Duddon (Bradley 1975) and recently in the Humber Es- tuary (Bratton, personal communication) — all in Britain, Penzé Estuary, Brittany and Orne Estuary, Normandy on the Atlantic coast of France (Ginsburger- Vogel 1991) and four locations (Goeree, Philipsland, Mastgat, Schouwen) in the Deltaic area of the Netherlands (Stock 1995). The distribution (Fig. 2) likely represents an incomplete pic- ture because of the difficulty in finding O. aestuarensis in the restricted area of occurrence in part of the mesohaline section of estuaries (see below). No records of occurrence have been found outside estuaries. 40°E 20°E Figure 1. Geographical distribution of O. mediterranea, from Wildish (1969). zse.pensoft.net 800 60°N 20°N 20°W 0° Wildish, D.J. & Radulovici, A.E.: Amphipods in estuaries 40°E 20°E Figure 2. Geographical distribution of O. aestuarensis for sampling locations known to 2019. To date, the British estuaries, Tamar, Medway and Duddon are the only ones where comprehensive estuarine distribution data has been collected. Results are summa- rised in Wildish (1987) and show that, in the Medway and Tamar, the most landward populations of O. aestua- rensis have female-biased sex ratios and few intersexes, whereas in the most seaward population, intersexes are common. In the high eulittoral, both species occur in dis- crete colonies separated by natural barriers with respect to estuarine penetration, such as soft mud or quay walls, which are unsuitable substrates. In the Tamar Estuary the most seaward O. aestuarensis consisted of a mixed pop- ulation with O. mediterranea and/or hybrids between the two. Mixed populations were not found in the Medway and Duddon estuaries. Similar estuarine penetration data were not collected for the French estuaries studied by Ginsburger- Vogel (1991), but in the one location sampled zse.pensoft.net in each of the Penzé and Orne estuaries, this author de- scribed mixed species populations. Mixed species populations occur only at the interface between O. aestuarensis and O. mediterranea distribution in the Tamar Estuary. Landwards of this interface location, only O. aestuarensis occurs and seaward only O. mediter- ranea. This distribution is interpreted to be governed by a low salinity switch at a location in the estuary where both species can co-exist. In the Medway Estuary, O. aestuaren- sis 1S present from above the Medway bridge, up-estuary for ~2.5 km (approximately one half of the mesohalinicum). Estuarine penetration terminates in the Medway where the highwater salinity is 31% of seawater (Fig. 3). O. mediter- ranea replaces O. aestuarensis below the Medway bridge where the salinity is 52% seawater and 1s present throughout the rest of the Estuary, into the Thames and North Sea/Strait of Dover, where suitable substrates and beaches are present. Zoosyst. Evol. 96 (2) 2020, 797-805 polyhalinicum Rochester bridge Medway bridge 801 Figure 3. Distribution of O. aestuarensis (closed circles) and O. mediterranea (open circles) in the Medway Estuary, from Wildish (1969). Taxonomic similarity between O. aestuarensis and O. mediterranea Morphologically, O. aestuarensis is close to O. mediterranea, although it can be distinguished by a number of minor taxonomic characters (Wildish 1987). Some examples include: male gnathopod 2 propodus palm with a proximal notch in aestuarensis, which is absent in mediterranea, male gnathopod 2 hinge tooth near the propodus articulation in mediterranea which 1s absent in aestuarensis; fewer third uropod peduncle spines and second antenna flagellum segments in aestuarensis (both are allometric characters). If a large population sample is available, O. aestuarensis can be recognised by the presence of a female-biased sex ratio and the occurrence of intersexes, versus a balanced sex ratio and few or no intersexes in O. mediterranea. Dorsal epidermal pigment patterns are similar in both species, except that O. aestuarensis has two elliptical holes in the solid mid- dorsal line which lack pigment in each body segment. O. mediterranea lacks the pigment perforations in the mid- dorsal line. Pigment pattern differences are recognised in the field, but can be best seen with the aid of a binocular microscope after preservation in formalin-based solution. Reproductive isolation Wildish (1970b) and Ginsburger-Vogel (1991) show that hybridisation between O. aestuarensis and O. mediter- ranea does occur in laboratory cultures. Whether hybri- disation occurs in the wild at mixed-species locations in estuaries 1s uncertain and requires molecular genetic stud- ies for confirmation. Both hybridisation experiments were conducted without considering the effect of salinity and temperature on phenotypic expression. In gammarids, sa- linity and temperature in conjunction with microsporidian endoparasites, are known to influence the occurrence of intersexes and sex ratio bias in the host population (Buln- heim 1978). The culture salinity was not stated during hy- zse.pensoft.net 802 bridisation experiments (Wildish 1970b); they were con- ducted with undiluted locally available seawater of Cl = 16.2%o0 (84% of 35%o salinity seawater) at 20-24 °C. The salinity and temperature during the hybridisation experi- ments conducted by Ginsburger- Vogel (1991) was also not stated, but it is assumed that relatively high salinity sea- water was used. Due to (1) the uncertainty regarding the salinity of seawater employed during culture and (2) the unrealistically high salinities probably used, the hybridisa- tion experiments should be repeated at appropriate salinity and temperature conditions for the estuary studied. The hybridisation studies in the Medway Estuary sug- gest that reproductive isolation between the two species is incomplete. The source of the two populations used in the British hybridisation experiments were ~8 km apart along the estuarine gradient: the most landward colony sampled was in the mesohalinicum and the most seaward in the polyhalinicum (Wildish 1970b). A physiological method of reproductive isolation is unnecessary if mechanisms 1 or 2 (see section on low salinity switch hypothesis) were involved in the origin of O. aestuarensis in estuaries. This is because the low salinity switch operates to exclude hy- bridisation. If dispersed landwards, O. mediterranea fe- males, at or downstream of the mixed interfacial location, would be unable to produce broods successfully because of the low salinity conditions (Wildish 1970b). Male O. mediterranea landward immigrants from the same down- stream locations, which can survive and presumably breed in low salinities, were unable to produce a viable brood when mated with a female O. aestuarensis (Wild- ish 1970b). Regarding seaward dispersal of O. aestuaren- sis, the males when mated with O. mediterranea females, were able to produce broods. Presumably, this is a way of maintaining the rare allele or gene within the genotype of O. mediterranea. Physiological ecology Species of Orchestia are sensitive to salinity conditions in estuaries and in lowland estuaries the high tide salinity may represent the salinity distribution limits (Table 1). Within the Medway Estuary, O. gammarellus is the least sensitive to low salinity and penetrates to the oligohalini- cum where the high-water salinity is ~ 5% of full-strength seawater. The sparse populations of O. gammarellus in the 5—10% seawater section of the estuary are likely Wildish, D.J. & Radulovici, A.E.: Amphipods in estuaries non-breeding immigrants dispersed from lower in the es- tuary (breeding absent in this part of the estuary, Morritt and Stevenson 1993). Experiments with diluted seawater and O. mediterra- nea showed that lethality (lethal concentration at which 50% of those tested die, LC50) was dependent on the in- ter-moult stage, with a wide range of 48-hour LC50s from 2 -15% of 35 ppt seawater (Wildish 1970a). The pre-moult stage was most susceptible to low salinity. These results suggest that direct lethality was not the primary cause of limited landward penetration in the estuary. Preliminary experiments to determine the effect of dilute seawater on fertility of O. mediterranea were conducted by Wildish (1970 a) with three seawater dilutions (14, 42 and 84% of 35 ppt seawater). Dilute salinity affected the inter-moult stage timing in breeding females, which increased the time between broods, as dilution increased. Both this and a loss of eggs and selective female mortality at low sa- linities reduced population fertility in low salinity condi- tions. These effects were present at both 14 and 42%, but not at 84% seawater (Wildish 1970a). Great strides have been achieved in understanding the physiology of ionic and osmotic regulation in talitrids (Morritt and Spicer 1998). In vitro-cultured, early em- bryos of O. gammarellus were killed at salinities below 40% seawater, yet if eggs developed in the marsupium (= brood pouch) of a living female, normal broods were produced at salinities down to 10% seawater (Morritt and Stevenson 1993). The explanation for these apparently conflicting results was that female O. gammarellus were able to control the osmotic concentration of the marsupial fluid, thereby allowing egg development at lower salini- ties (Morritt and Spicer 1998). Similar experiments have not been conducted with O. mediterranea or O. aestu- arensis, but it is proposed that marsupial fluid control was absent in the former species. This 1s consistent with the preliminary fertility experiments with O. mediter- ranea (Wildish 1970a) and that this species depends on frequent tidal wetting to maintain the marsupial fluid os- mo-concentration. Further experiments with both species are clearly needed to clarify this. O. aestuarensis, which extends into the Medway Estuary at lower salinities than its sibling, 1s proposed to have developed a rudimenta- ry form of marsupial fluid control that allows successful broods to be produced down to salinities of 31% seawater. O. mediterranea can only produce broods consistently in salinities > 52% seawater. Table 1. Distribution limits at high water of Orchestia in two lowland estuaries: Deltaic Area, The Netherlands (Den Hartog 1963b) and Medway Estuary, UK (Wildish 1987). The chlorinity values (parts per thousand) used in both reports have been converted to salinity (parts per thousand) by multiplying by 1.80655. Species Deltaic Area Salinity (ppt) Percent of full-strength seawater (S = 35ppt) Medway estuary Percent of full-strength seawater (S = 35ppt) Salinity (ppt) O. gammarellus 3.25 > 29.81 9.3-100 1.81 > 29.81 5.2-100 O. mediterranea 9.03 > 29.81 25.8-100 18.07 > 29.81 51.6-100 O. aestuarensis ? ? 10.84-18.07 31-51.6 zse.pensoft.net Zoosyst. Evol. 96 (2) 2020, 797-805 Genetic distance between siblings The Barcode of Life Data System (BOLD, wwwboldsys- tems.org, Ratnasingham and Herbert 2007) was searched for published and publicly available mitochondrial CO1 sequences of Orchestia in May 2020. The only oligoha- line species, O. aestuarensis (N = 37) was compared with all the euryhaline Orchestia species. The closest species was O. mediterranea (N = 24), its sibling, with a mean CO1 genetic distance of 10.2%. The other mean inter- specific genetic values were much higher: 16% with O. montagui Audouin, 1826 (N = 80), 19.8% with O. gam- marellus (Pallas, 1766) (N = 244) and 21.0% with O. ste- phenseni Cecchini, 1928 (N = 3) Geological background The importance of the geological background in the study of O. mediterranea in estuaries of the coasts of the northeast Atlantic and Mediterranean Sea is that it provides a times- cale for the evolution of its sibling species O. aestuarensis. The Pleistocene history of the Thames and Medway estuaries in Britain are discussed by Bridgland and D’OI- ier (1995). None of the glacials, including the last, the Devensian (115,000 to 11,700 years BP), reached the southern estu- aries (e.g. Medway, Tamar) in Britain (Lee 2011), which suggests an estuarine fauna that is at least 400,000 years old. By contrast, the Duddon Estuary in the Lake District of north-western and the Humber Estuary in north-east- ern Britain was ice-covered during the Devensian glacial (Lee 2011) and its fauna therefore extirpated, with a re- placement age of < 10,000 years. Discussion O. aestuarensis/O. mediterranea sibling Species pair in estuaries All three of the proposed mechanisms of in situ evolu- tion of O. aestuarensis from O. mediterranea in estuaries avoid the need for natural dispersal by the low salinity sibling from one estuary to another. Hypothetical mech- anism 3 involves de novo evolution in each new estuary colonised by O. mediterranea. It seems to be less likely in view of the phenotypic similarities found in the few populations so far studied. The oligohaline O. aestuaren- sis in northern Britain would have diverged more recently (< 10,000 years) than southern species (> 400,000 years). Population divergence estimates, based on molecular genetic methods, such as the mitochondrial COlgene (Knowlton and Weight 1998), could indicate a temporal difference in the date of origin. Results expected would be for similar ages from different geographical samples, if mechanisms | and 2 were operating and variably di- vergent times for each estuary if mechanism 3 were op- 803 erating. For the latter, northern populations would have diverged more recently than southern ones. For the ability to breed at low salinity (< 52% seawa- ter), we propose that a dimorphic allele or linked set of alleles is selected for by low salinity conditions, a gene(s) is/are switched on by low salinity conditions or allopatric or parapatric speciation occurs 1n each new estuary, colo- nised by O. mediterranea. The minor morphological and dorsal pigment pattern differences with O. mediterranea in O. aestuarensis (Wildish 1987) are linked to genes or alleles governing the ability to breed at low salinities. In a laboratory intrapopulation cross with males and females of O. mediterranea at Upnor Castle (polyhalini- cum), Wildish (1970b) found that a male and female and 11 intersexes were produced with aestuarensis pigmenta- tion patterns in the first generation, F, (N = 50). Ginsburg- er-Vogel (1991) also found a form of O. mediterranea in a Siyjean salt marsh, on the French Mediterranean coast, which sometimes spontaneously produced the “aestu- arensis” dorsal pigment pattern phenotype. Both obser- vations suggest that O. mediterranea does carry alleles or genes characteristic of O. aestuarensis, supportive of either mechanism 1 or 2. Histological observations and grafting experiments undertaken by Ginsburger- Vogel (1991) showed that 1n- tersexuality and female-biased sex ratios, as occur in wild populations of O. aestuarensis, are caused by the physi- ological activities of microsporidians within the host. We suggest that the relationship between the microsporidi- an(s) and its oligohaline amphipod host are symbiotic. This is because the microsporidian is provided with food and lodging by the host, but in return — perhaps by secret- ing appropriate hormone mimics — changes genetic males to females. Female biasing increases the fertility of a giv- en population (Wildish 1971). The selective advantage here is not to the individual, but to the population. Such an adaptation may be of importance to small, precarious populations isolated in estuaries, like O. aestuarensis. In an earlier paper (Wildish 1987), the genetic mecha- nism hypothesised to account for the evolution of O. aestu- arensis included a superior ability to survive and breed in lower salinity conditions which was linked to the occur- rence of intersexes/female bias. Ginsburger- Vogel (1991) showed that the occurrence of intersexes/female bias in O. aestuarensis is not linked to the ability to breed in low salinities, but independently results from co-evolution of an endosymbiotic protozoan and its host, O. aestuarensis. The enduring commonality between the two evolutionary mechanisms is that both are controlled by the same salini- ty switch. This leaves superior ability to breed and female biasing in low salinity conditions as two independent se- lective factors in the evolution of O. aestuarensis. The genetic changes hypothesised to underlie low sa- linity switching in Orchestia occurred in the geological (recent) past, likely in different environments than those of today. It is therefore problematic to use direct experi- mentation to test the natural selection hypothesised to be involved. Instead, we have proposed inductive inference zse.pensoft.net 804 Wildish, D.J. & Radulovici, A.E.: Amphipods in estuaries Table 2. Putative sibling species pairs of Gammaridae associated with estuaries from the North Atlantic Ocean. Taxonomic names as in World Register of Marine Species (WoRMS), accessed April 2019, CO1 data from BOLD accessed May 2020, N = number of oligohaline/euryhaline individuals. Estuary Putative Sibling Species Pair Reference Mean % COl N Brackish / estuarine (oligohaline) Marin e/ Estuarine (euryhaline) genetic distance Canadian Gammarus tigrinus Sexton, 1939 Gammarus lawrencianus Bousfield, 1956 Steele and Steele 24.86 172/70 estuaries (1991) North American Gammarus daiberi Bousfield, 1969 Gammarus tigrinus Sexton, 1939 Bousfield (1973) 14.54 4/172 estuaries Deltaic Area, Gammarus zaddachi Sexton,1912 Gammarus salinus Spooner, 1947 Den Hartog (1964) 14.62 60/82 Holland Deltaic Area, Gammarus salinus Spooner, 1947 Gammarus locusta (Linnaeus, 1758) Den Hartog (1964) 26.35 82/94 Holland Deltaic Area, Echinogammarus marinus (Leach, 1815) Echinogammarus obtusatus (Dahl, 1938) | Den Hartog (1964) 33.29 36/31 Holland methods to do this. Strong evidence in support of low salinity switching will be provided if all the individual criteria, zoogeographic, physiological, ecological and ge- netic, reviewed above, support it. Amphipod sibling species pairs in estuaries: a possible general salinity switch hypothesis Other species of amphipods are known to be sensitive to salinity conditions and some are confined to estuar- ies as sibling species (e.g. some gammarids) (Table 2). Amongst the Gammaridae of the North Atlantic region, we found five pairs of potential sibling species in estuar- ies and obtained their mitochondrial CO1 sequences from BOLD and compared interspecific genetic distances, as in Orchestia. Two. species pairs, G. zaddachi/G. salinus and G. daiberi/G. tigrinus, were found to be genetically closer to each other than to other congeners (Table 2) and, thus, they are putative candidates for evolution by salin- ity switching. For two other species pairs of Gammarus and one pair of Echinogammarus (Table 2), there was no evidence that they were closer genetically than others within the same genus. Consequently, they are eliminat- ed as candidates for evolution by salinity switching. We recognise that the genetic test we have used herein is not definitive on its own, because evolutionary pathways of species with reduced genetic distance between siblings could be directed by physical or biological factors other than salinity. Genetic relatedness and ecological data (that the oligo- haline amphipod is limited to low salinities in estuaries), together with other related biological data, are suggestive that low salinity conditions directed the evolution in these amphipods. This study illustrates that limited ecological/genetic data are useful in identifying pairs of sibling species of estuarine amphipods which may have evolved by salinity switching. Using this method, three sibling species pairs were identified: O.aestuarensis/O. mediterranea, G. zaddachi/G. salinus and G. daiberi/G. tigrinus. Further studies, based on the six biological/geological criteria used herein to infer salinity switch evolution in O.aestuarensis/O. mediterranea, could zse.pensoft.net be applied to the two selected Gammarus sp. siblings. The location of a gene(s) for salinity switching would ultimately confirm the hypothesis. We suspect that there will be other sibling species pairs of amphipods which have evolved in this way in other es- tuaries throughout the world and that our study is a step forward in shedding light on estuarine amphipod evolution. Acknowledgements We thank Ms. Enid Bradley and Mr. John Bratton for sharing their discoveries of O. aestuarensis in the Duddon and Humber estuaries, respectively and a reviewer for improving an earlier version. Thanks to the Museum fir Naturkunde, Berlin for supporting the publication costs. References Arfianti T, Wilson S, Costello MJ (2018) Progress in the discovery of amphipod crustaceans. PeerJ. 6: e5187. https://doi.org/10.7717/ peerj.5187 Bousfield EL (1973) Shallow-Water Gammaridean Amphipoda of New England, Cornell University Press, New York, 312 pp. Bradley E (1975) The biology of Orchestia mediterranea A. Costa and Orchestia gammarella (Pallas) in salt marshes of the Duddon estu- ary. M.Sc. Thesis, University of Lancaster, U.K. Bridgland DR, D’Olier B (1995) The Pleistocene evolution of the Thames and Rhine drainage systems in the southern North Sea ba- sin. In: Preeca, RC (Ed) Island Britain: A Quarternary Perspective. Geological Society Special Publication No. 96: 27-45. https://doi. org/10.1144/GSL.SP.1995.096.01.04 Bulger AJ, Hayden BP, Monaco ME, Nelson DM, McCormick MG (1993) Biologically based estuarine salinity zones derived from multivariate analysis. Estuaries 16: 311-322. https://doi. org/10.2307/1352504 Bulnheim H-P (1978) Interaction between genetic, external, and para- sitic factors in sex determination of the crustacean amphipod Gam- marus duebeni. Helgoland Wiss Meeresunters 31: 1-33. https://doi. org/10.1007/BF02296989 Dahl E (1967) Land amphipods from the Azores and Madeira. Boletin do Museu municipal do Funchal 21: 8-23. Zoosyst. Evol. 96 (2) 2020, 797-805 Den Hartog C (1960) Comments on the Venice-System for the classifi- cation of brackish waters. Internationale Revue der gesmaten Hyd- robiolgie und Hydrographie 45: 481-485. Den Hartog C (1963a) The amphipods of the Deltaic region of the rivers Rhine, Meuse, and Scheldt in relation to the hydrography of the area. Part I. Introduction and Hydrography. Netherlands Journal of Sea Research 2: 29-39. https://doi.org/10.1016/0077-7579(63)90003-6 Den Hartog C (1963b) The amphipods of the Deltaic region of the riv- ers Rhine, Meuse, and Scheldt in relation to the hydrography of the area. Part II. The Talitridae. Netherlands Journal of Sea Research 2: 40-67. https://doi.org/10.1016/0077-7579(63)90004-8 Den Hartog C (1964) The amphipods of the Deltaic region of the rivers Rhine, Meuse, and Scheldt in relation to the hydrography of the area. Part II]. The Gammaridae. Netherlands Journal of Sea Research 2: 407-457. https://doi.org/10.1016/0077-7579(64)90002-X Ginsburger-Vogel T (1991) Intersexuality in Orchestia mediterranea Costa, 1853, and Orchestia aestuarensis Wildish, 1987 (Amphipo- da): a consequence of hybridization or parasitic infestation? Journal of Crustacean Biology 11: 530-539. https://do1.org/10.2307/1548522 Knowlton N, Weight LA (1998) New dates and new rates for divergence across the isthmus of Panama. Proceedings Royal Society of London, Series B, 265: 2257-2263. https://do1.org/10.1098/rspb.1998.0568 Kolding S (1985) Genetic adaptation to local habitats and speciation processes within the genus Gammarus (Amphipoda: Crustacea). Marine Biology 89: 249-255. https://doi.org/10.1007/BF00393658 Horton T, Lowry J, De Broyer C, Bellan-Santini D, Coleman CO, Cor- bari L, Costello MJ, Daneliya, M, Dauvin J-C, Fi8er C, Gasca R, Grabowski M, Guerra-Garcia JM, Hendrycks E, Hughes L, Jaume D, Jazdzewski K, Kim Y-H, King R, Krapp-Schickel T, LeCroy S, Lorz A-N, Mamos T, Senna AR, Serejo C, Sket B, Souza-Filho JF, Tandberg AH, Thomas JD, Thurston M, Vader W, Vainola R, Vonk R, White K, Zeidler W (2020) World Amphipoda Database. http:// www.inarinespecies.org/amphipoda [on 2020-06-25] Lee JR (2011) Cool Britannia — from Milankovich wobbles to Ice Ages. Mercian Geologist 17: 274-279. Morritt D, Stevenson TDI (1993) Factors influencing breeding initiation in the beachflea Orchestia gammarellus (Pallas) (Crustacea: Am- phipoda). Journal Experimental Marine Biology and Ecology 165: 191-208. https://doi.org/10.1016/0022-0981(93)90105-W Morritt D, Spicer JI (1998) The physiological ecology of talitrid am- phipods: an update. Canadian Journal of Zoology 76: 1965-1982. https://doi.org/10.1139/z98-168 805 Odum EP (1971) Fundamentals of Ecology. Third Ed. W. B. Saunders Co., Philadelphia. 574 pp. Ratnasingham S, Hebert PDN (2007) BOLD: The Barcode of Life Data System (http://www. barcodinglife.org). Molecular Ecology Notes 7: 355-364. https://doi.org/10.1111/j.1471-8286.2007.01678.x Ruffo S (1938a) Studi sui Crostacei Anfipodi. Gli Anfipodi del Medi- terranea. Annali del Museo civico di storia naturale di Genova 60: 127-151. Ruffo S (1938b) Studi sui Crostacei Anfipodi. Gli Anfipodi del Mar Rosso. Annali del Museo civico di storia naturale di Genova 60: 152-180. Schellenberg A (1942) Flohkrebse oder Amphipoda. Tierwelt Deutsch- lands 40: 1-252. Steele DH, Steele VJ (1991) Effects of salinity on the survival, growth rate, and reproductive output of Gammarus lawrencianus (Crus- tacea, Amphipoda). Marine Ecology Progress Series 78: 49-56. https://doi.org/10.3354/meps078049 Stock JH (1995) Orchestia aestuarensis (Amphipoda, Talitridae) nu ook in Nederland Aangetroffen. Het Zeepaard 55(4): 81-84. West-Eberhard MJ (1989) Phenotypic plasticity and the origins of di- versity. Annual Review of Ecology, Evolution, and Systematics 20: 249-278. https://doi.org/10.1146/annurev.es.20.110189.001341 Whitman DW, Agrawal AA (2000) What is phenotypic plasticity and why is it important? In: DeWitt T, Scheiner S (Eds) Phenotypic Plas- ticity, Oxford University Press, Oxford, 65 pp. Wildish DJ (1969) Studies on taxonomy, ecology, and behavior of Orchestia gammarella (Pallas), O. mediterranea A. Costa and O. cavimana Heller in the Medway estuary. Ph.D. Thesis, University of London, 252 pp. Wildish DJ (1970a) Some factors affecting the distribution of Orchestia (Crustacea, Amphipoda) in estuaries. Journal Experimental Marine Biology and Ecology 5: 276-284. https://doi.org/10.1016/0022- 0981(70)90007-9 Wildish DJ (1970b) Polymorphism in ranea. A. Costa. Crustaceana 19: org/10.1163/156854070X00446 Wildish DJ (1971) Adaptive significance of a biased sex ratio in Orches- tia. Nature 233: 54—55. https://doi.org/10.1038/233054a0 Wildish DJ (1987) Estuarine species of Orchestia (Crustacea: Amphi- poda: Talitroidea) from Britain. Journal Marine Biological Associ- ation of the United Kingdom 67: 571-583. https://doi.org/10.1017/ $00253 15400027302 mediter- https://doi. Orchestia 113-118. zse.pensoft.net