Genetic hypervariability of a Northeastern Atlantic venomous rockfish

Introduction

Understanding the interplay between historical and current climate changes in shaping genetic diversity is pivotal to infer future changes in the distribution of marine species. Climate warming is expected to significantly impact marine organisms with range disturbances anticipated (e.g., Cheung et al., 2009; Lasram et al., 2010; Hollowed et al., 2013; Poloczanska et al., 2014; Lenoire & Svenning, 2015; García Molinos et al., 2016). Indeed, climate-induced contractions, expansions, shifts and population extirpations across distribution areas have been amply recorded (e.g., Nicastro et al., 2013; Yeruham et al., 2015; Yu & Chen, 2018; Aguilera et al., 2020). In the past three decades, several advances in molecular and analytical tools led to the accumulation of data on the genetic diversity of marine organisms (e.g., Helyar et al., 2011; Plough, 2016; Allendorf, 2017) and connectivity (for reviews see Selkoe et al., 2016; Bryan-Brown et al., 2017). Different phylogeographic patterns have been recorded in the North-eastern Atlantic, ranging from panmictic species with homogeneous genetic diversity throughout their distribution range to species with marked population differentiation (e.g., Francisco et al., 2011; Jenkins, Castilho & Stevens, 2018).

Traditionally, the degree of genetic connectivity has been related to contemporary and historical factors that combined shape the present-day observed patterns (e.g., Gaggiotti et al., 2009; Woodall, Koldewey & Shaw, 2011; Reis-Santos et al., 2018). Marine species’ geographical spread and population differentiation may be influenced by their potential for dispersal and gene flow, depending on passive larval dispersal and active adult migratory movements (e.g., Selkoe & Toonen, 2011; Pascual et al., 2017). The interaction between life-history traits such as pelagic larval duration (PLD), larval behaviour and swimming abilities, post-settlement processes and oceanographic regimes play a role in the dispersal range of species with low adult dispersal ability (e.g., Bowen et al., 2006; Galarza et al., 2009; Schunter et al., 2011; Dalongeville et al., 2015) (see Bryan-Brown et al. (2017) for a review). However, there is still an ongoing debate on the reliability of using a species’ PLD as a proxy for population connectivity (e.g., Bradbury et al., 2008; Weersing & Toonen, 2009).

Amongst the historical factors shaping present-day connectivity patterns are climate changes associated with the Pleistocene glacial cycles, geological barriers, salinity gradients, hydrodynamics and paleoecological history (e.g., Olsen et al., 2004; Bigg et al., 2008; Palero et al., 2008). These very dynamic factors are recurrently invoked to explain the biogeography of marine organisms (e.g., Jenkins, Castilho & Stevens, 2018). At the peak of the last glacial maximum (LGM), around 21 thousand years ago (kya), the sea level dropped by 110–150 m (Lambeck & Purcell, 2005). Still, the connection between the Mediterranean and the adjacent Atlantic waters was not interrupted (Flores et al., 1997), sustaining glacial refugia hypothesised within this basin (Maggs et al., 2008).

The Atlantic-Mediterranean phylogeographic break is well documented for several marine organisms (e.g., Magoulas et al., 2006; Taboada & Pérez-Portela, 2016), notwithstanding the reopening of the Gibraltar Strait connection at the end of the Messinian Salinity Crisis (~5.33 Mya) (Hsü, Ryan & Cita, 1973; Krijgsman et al., 1999; Duggen et al., 2003). For others, often closely related species, the gene flow is unconstrained (e.g., Stamatis et al., 2004; Castilho et al., 2017; Lourenço et al., 2017) (see Patarnello, Volckaert & Castilho, 2007 and Kettle et al., 2011 for reviews). For the former, the inferred barrier has been associated with hydrological conditions that prevent migration either in recent times or in the Pleistocene, or both. This soft barrier location, however, is not consistent and for some taxa, the observed phylogeographic break is at the Strait of Gibraltar in the entrance of the Mediterranean basin (e.g., Sala-Bozano, Ketmaier & Mariani, 2009; Fruciano et al., 2011; García-Merchán et al., 2012), whilst for several others is further East, at the Almeria-Oran front in the Alboran Sea region (e.g., Zane et al., 2000; Lemaire, Versini & Bonhomme, 2005; Viñas et al., 2014).

One of the evident effects of climate change is the increase in seawater temperature, which translates into a global tropicalization trend (e.g., Bianchi & Morri, 2003; Wernberg et al., 2016) that also affects the Northeastern Atlantic. Along the southern and western coasts of the Iberian Peninsula, several organisms are expanding their poleward distribution, some with great impact in community composition (e.g., Lourenço et al., 2012; Nicastro et al., 2013; Bode et al., 2020; Robalo et al., 2020). One of the fish species recently reported off southwestern continental Portugal is the Madeira rockfish, Scorpaena maderensis Valenciennes 1833 (Encarnação et al., 2019), an estimated moderately vulnerable species (36/100) according to the model by Cheung, Pitcher & Pauly (2005). The IUCN Red List highlights the unknown current population trend of this least concerned species (Nunoo et al., 2015). Its genetic characterization and phylogeography have therefore become imperative.

The Madeira rockfish, Scorpaena maderensis, is distributed in the eastern Atlantic, including the islands of Azores, Madeira, Canaries and Cape Verde and in the Mediterranean Sea (Hureau & Litvinenko, 1986; Eschmeyer et al., 1990; Froese & Pauly, 2019). The species is a benthic sedentary species, mostly occupying shallow coastal areas with rocky bottoms and estuaries (usually underneath boulders or in crevices). Congeneric species, S. scrofa and S. porcus, have high residency and narrow home ranges (Özgül et al., 2019). Based on the present-day known distribution, mainly of subtropical nature, the estimated seawater temperature range for the species is 16–25 °C (Kaschner et al., 2019), but there are no studies on the thermal tolerance of the species. The Madeira rockfish is a generalized and opportunistic feeder of benthic or epibenthic crustaceans and, occasionally, algae, gastropods, polychaetes and small fishes (La Mesa, La Mesa & Tomassetti, 2007), and shows sexual dimorphism in growth rate, maximum size and longevity, with differences registered between the Mediterranean and Azorean populations (Morato et al., 2001; La Mesa, La Mesa & Micalizzi, 2005). A specialized mode of oviparity is described for the genus and the eggs are deposited as a whole in a protective gelatinous matrix that facilitates spawning cohesiveness and floatation (Wourms & Lombardi, 1992). The spawning season takes place from December to February in the Mediterranean (La Mesa, La Mesa & Micalizzi, 2005) and from March to June in the Azores (Costa, 2007). However, structural features of its biology are yet to be clarified, particularly the ones related to reproduction and early-life traits.

The present work is the first population study for this species, comprising a wide sampling coverage of its distribution range (seven locations from the Atlantic and the Mediterranean Sea), and two molecular markers (mitochondrial and nuclear) to screen the genetic diversity of S. maderensis with the following objectives: (1) evaluate the genetic diversity within and among locations; (2) assess the population genetic structure of the Madeira rockfish; and (3) evaluate the putative existence of a soft barrier between the Atlantic and the Mediterranean populations.

Materials and Methods

Sampling

Specimens of S. maderensis were collected from seven locations across its distributional range in the Atlantic and Mediterranean: Cyprus (CYP), Greece (GRE—Euboea), Sicily (SIC—Messina, Riposto and Siracusa; Italy), Azores (AZO—Faial; Portugal), Madeira (MAD—Funchal; Portugal), Selvagens (SEL; Portugal) and Canaries (CAN—Tenerife; Spain) (Fig.1 and Table 1). Specimens were provided by fishers as the species is a frequent by-catch in coastal short-range artisanal fisheries and fins were clipped after assessing the species identification for each individual. Samples were preserved in 96% ethanol and deposited in ISPA-IU/MARE tissue collection.

Table 1:

Sample locations, coordinates, sizes and summary statistics for the mitochondrial control region and the nuclear S7 gene of Scorpaena maderensis.

Location	Code	Long	Lat	Mitochondrial Control Region									Nuclear S7
				N	nH	nP	nS	nP/H	h	π (%)	R	pR	N	nH	nP	nS	nP/H	h	π (%)	R	pR
Cyprus	CYP	35.091	33.344	13	12	9	11	75.00%	0.987	2.12	9.733	6.166	22	22	19	22	86.36%	1.000	3.05	19.209	9.714
Greece	GRE	37.740	24.059	7	7	7	7	100.00%	1.000	3.66	7.000	5.503	14	12	10	10	83.33%	0.978	3.42	12.000	5.553
Sicily	SIC>	37.708	15.202	42	31	19	22	61.29%	0.984	2.50	11.741	5.780	22	22	20	22	90.91%	1.000	2.93	19.209	10.032
Mediterranean	MED	–	–	62	47	37	36	78.72%	0.989	2.58	12.445	3.872	58	54	50	50	92.59%	0.998	3.05	24.002	2.130
Canaries	CAN	28.47	−16.255	22	19	11	16	57.89%	0.987	2.77	11.037	6.053	44	40	32	37	80.00%	0.995	3.05	22.381	15.128
Selvagens	SEL	30.108	−15.957	20	20	15	20	75.00%	1.000	3.02	11.667	8.097	48	47	39	46	82.98%	0.999	2.92	23.775	16.675
Madeira	MAD	32.694	−16.775	20	20	17	20	85.00%	1.000	3.28	11.667	8.629	32	27	18	22	66.67%	0.990	3.13	19.662	10.923
Azores	AZO	38.522	−28716	22	19	12	16	63.16%	0.987	2.79	11.037	4.215	38	32	25	28	78.13%	0.989	3.04	20.502	12.814
Atlantic	ATL	–	–	84	68	58	57	85.29%	0.993	3.01	12.897	4.215	162	127	123	113	96.85%	0.992	2.87	24.752	8.478
Total	–	–	–	146	105	–	81	–	0.989	2.87	–	–	220	177	–	159	–	0.995	3.60	–	–

DOI: 10.7717/peerj.11730/table-1

Note:

Long, longitude; Lat, latitude; N, number of gene copies; n_H, number of haplotypes; n_P, number of private haplotypes; n_S, singletons; n_P/H, proportion of private haplotypes; h, haplotype diversity; π, nucleotide diversity; R, allelic richness; pR, private allelic richness.

Results

For the CR, a fragment of 354 bp was amplified and the 146 sequences obtained defined 105 haplotypes, with a total of 80 polymorphic sites found. Differences among haplotypes corresponded to 80 transitions, 12 transversions and 1 indel. For the S7 the 220 sequences (110 individuals) defined a total of 177 haplotypes. For this marker, the fragment obtained was 517 bp long and the differences among haplotypes corresponded to 206 mutations (70 transitions, 80 transversions and 50 indels). The S. maderensis S7 dataset, as a whole, conformed to the HWE (p = 0.998), although 36 out of the 171 polymorphic sites were in heterozygote deficit. For both fragments, the genetic diversity indices were generally very high, with little variation among collection sites (Table 1). The proportion of private haplotypes was high for all the locations (Table 1), with only 9.52% and 2.26% being shared between the Atlantic and the Mediterranean, for the CR and the S7, respectively.

The obtained haplotype networks revealed deep hyper-diverse bush-like genealogies, with a large number of singletons, few shared haplotypes and no evidence for geographic structure (CR: Fig. 2, S7: Fig. 3; see also Table 1, details on haplotype composition are given in Table S1 in Supplementals Materials).

The divergence parameters yielded significant values for the overall CR (Table 2). In both markers, results from the pairwise comparisons were equivocal, with F_ST showing non-significant values, Nei’s G_ST revealing significant values for some of the comparisons, and Hedrick’s G’_ST and Jost’s D yielding all comparisons statistically significant (Table 2), i.e., all pairs of sampling sites usually have distinct haplotypes. In fact, eight (CR and S7) out of 22 pairwise comparisons revealed complete haplotypic differentiation (D = 1), including between some of the geographically closest sampling sites (Table 2). This high structuring was supported by the AMOVA results (CR: F_ST = 0.031, p = 0.004; S7: F_ST = 0.016, p = 0.005), which also revealed that variation among sampling sites accounted for only 3.08% (CR) and 1.58% (S7) of the total variation (Table S2 in Supplemental Materials).

Discussion

The present work comprises a wide geographic sampling coverage of Scorpaena maderensis with locations from the Atlantic and the Mediterranean Sea and a molecular dataset with two markers. Our results highlight two main features in the population genetics of the Madeira rockfish: (1) deep hyper-diverse bush-like genealogies, characterised by large numbers of singletons and few shared haplotypes; and (2) absence of genetic structure across sampled locations, with no detectable Atlantic-Mediterranean break in connectivity. Before discussing these findings in detail, we address the main caveats concerning this study: the sampling strategy and the molecular markers used. Although most locations are represented by numbers of individuals in line with previous phylogeographic studies in marine species, one can a posteriori posit that the high number of singleton haplotypes found is biased by insufficient sampling. In fact, a recent study published by our team recorded even higher genetic diversity in a coastal fish species, revealing that it would be necessary to sample a total of 700 individuals for the sampling to be representative of the population (Robalo et al., 2020). Additionally, we have no samples from intermediate locations between the Atlantic archipelagos and the Western Mediterranean. These areas are not in the reported distribution of the species (Froese & Pauly, 2019) and the few reported individuals are occasional appearances (Encarnação et al., 2019). Another caveat is using only one mitochondrial and one nuclear marker in a day and age where next-generation sequencing producing thousands of markers are being increasingly used. This study is in line with previous research in the pursuit for patterns and processes involved in the phylogeography of the species from the North-East Atlantic (e.g., Bargelloni et al., 2005; Debes, Zachos & Hanel, 2008; Francisco et al., 2011; Robalo et al., 2013a). These previous studies used the same set of markers, allowing across species comparisons and multi-species approaches (e.g., Robalo et al., 2012; Robalo et al., 2013b; Francisco et al., 2014; Almada et al., 2017; Castilho et al., 2017) while revealing very distinct patterns.

Supplemental Information