How environmental change affects a species' phenotype is crucial
not only for taxonomy and biodiversity assessments but also for its
application as a palaeo-ecological and ecological indicator. Previous
investigations addressing the impact of the climate and hydrochemical regime
on ostracod valve morphology have yielded contrasting results. Frequently
identified ecological factors influencing carapace shape are salinity,
cation, sulfate concentrations, and alkalinity. Here, we present a thorough
approach integrating data with the carapace outline and surface details of
the ubiquitous Neotropical cytheroidean ostracod species
Understanding how species respond to environmental change is crucial for their application as proxies for past climate fluctuations as well as in forecasting the future dynamics and distributions of species. Morphological diversity represents a key feature of the interpretation of faunal changes (Wagner and Erwin, 2006) and ecological shifts (Mahler et al., 2010) and prompts discussions about speciation and extinction processes over time (e.g. Ciampaglio, 2004). Differences in shape and size among species have been shown to relate to changes of environmental parameter, in particular, differences in temperature across various clades (e.g. Loehr et al., 2010; Maan and Seehausen, 2011; Danner and Greenberg, 2015). Within freshwater invertebrates, ecophenotypic response has been documented for a variety of species, both recent and fossil (e.g. Hellberg et al., 2001; Zieritz and Aldridge, 2009; Inoue et al., 2013; Neubauer et al., 2013; Clewing et al., 2015).
Geographic overview of the sampled populations (modified from
Wrozyna et al., 2016). The label FL-LX
Ostracods represent a popular proxy group for climate and ecosystem changes due to their occurrence in various habitats, ranging from most inland waters to marine and interstitial and even semiterrestrial and terrestrial environments (e.g. Horne, 2005). Their distribution is controlled by ecological factors such as salinity, temperature, and ion composition of the ambient water (e.g. Ruiz et al., 2013). The study of ecophenotypic variation in response to environmental change (Anadón et al., 2002; Frenzel et al., 2012; Fürstenberg et al., 2015; van der Meeren et al., 2010) demonstrates another approach using ostracods for palaeoenvironmental studies. Due to their calcitic valves, they have an excellent fossil record and are utilized as palaeoenvironmental and biostratigraphic indicators (Anadón et al., 2002). A number of studies have shown that ornamentation, noding, sieve pore shape, and carapace size are linked to environmental factors, e.g. salinity, temperature, water depth, and nutrient availability (van Harten, 1975; Yin et al., 1999; Majoran et al., 2000; van Harten, 2000; Anadón et al., 2002; Frenzel and Boomer, 2005; Medley et al., 2007; Marco-Barba et al., 2013; Meyer et al., 2016; Boomer et al., 2017). Especially with the rise of morphometric techniques, investigations dealing with carapace shape variation in relation to environmental variables have also increased (Yin et al., 1999; Baltanas et al., 2002; van der Meeren et al., 2010; Ramos et al., 2017; Grossi et al., 2017). Yet, the use of morphological data, even those based on morphometric analyses (Baltanas et al., 2002, 2003; van der Meeren et al., 2010; Grossi et al., 2017), has been restricted to either landmark-based or outline-based studies but has rarely used a combination of both (e.g. Ramos et al., 2017). Few studies integrate geographic gradients into their statistical analyses and corresponding climate variables or a reduced number of predictor variables. Moreover, shape–environment relationships are commonly identified based on simple linear regressions or qualitative observations on multivariate ordination methods.
Here, we apply a thorough approach integrating morphometric data from
carapace outline and surface details, as well as several climatic and
hydrochemical variables, in order to investigate a potential link between
morphology and environmental conditions. The subject of study is the valves
of the Neotropical cytheroidean ostracod species,
Specimens of
Altogether, 15 variables were included in the analyses. Simultaneously to
water sampling, field variables (electrical conductivity, water temperature
and pH) were measured in situ at all sample sites using a WTW multi-sensor
probe (Multi 3420 Set C). Water samples were taken with plastic bottles,
promptly filtrated using a syringe filter with a filter pore size of
0.45
Valve morphology was captured using a combination of landmarks and semilandmarks. Eight landmarks (LMs) were chosen to characterize anterior pore tubules (LM 1–5, type I) and the dorsal dip point of the posterior curvature (LM 6, type II) as well as to delimitate maximum anterior and posterior curvatures (LM 7–8, type III). The carapace outline was defined by two curves between LM 7 and 8, each comprised of 30 equidistantly spaced semilandmarks (see also Wrozyna et al., 2016). All points were set on digitized SEM images using the program TpsDig v. 2.17 (Rohlf, 2013). The sliders file determining sliding direction of the semilandmarks during the Procrustes alignment was created in TpsUtil v. 1.58 (Rohlf, 2015). A generalized least-squares Procrustes analysis, computing consensus configuration, partial warps and relative warps (RWs), was performed using the program TpsRelw v. 1.65 (Rohlf, 2016). Thin-plate spline deformation grids were used to visualize deviations of selected configurations from the mean and to identify morphological characteristics that account for differences among geographic regions. For details on the method see Rohlf and Slice (1990) and Bookstein (1996).
We ran preliminary analyses for each dataset to identify major outliers that may bias the morphometric analyses by overemphasizing particular directions in the morphospace (and associated morphological characteristics). Such a distortion may severely impede the sound interpretation of follow-up statistical analyses.
In order to study the covariance between shape variation and environmental variables, two-block partial least-squares (PLS) analyses were performed using the software PAST 3.18 (Hammer et al., 2001). A big advantage over other ordination methods such as principal components analysis, this method disregards inter-block variation that may mask between-block covariance (Mitteroecker and Bookstein, 2008, 2011). Using all RWs in the PLS analysis might severely bias the pattern since, contrary to their descending significance in terms of explaining shape variation, they would be treated equally by the analysis. Therefore, we restricted the morphological block to RW 1–20, which account for at least 98.6 % of the total shape variations in all four datasets. The environmental variables were log10-transformed to constrain the orders of magnitude involved. PLS analyses were computed based on correlation matrices.
Relative warp analyses of left
The PLS analysis provides an idea of the overall strength of the relationships between shape and environment. To identify the parameters that affect specific morphological traits or combination of traits, multiple regression analyses were conducted on selected RWs in the statistical environment R v. 3.3.2 (R Core Team, 2016). Only warps (1) along which biogeographic differentiation was observed, (2) with an amount of shape variation higher than 10 % of the total variation, and (3) with PLS loading values higher than the mean loading value (based on absolute values) were considered. These selection criteria were chosen in order to prevent the misinterpretation of seemingly strong relationships between shape and environmental variables. Since the environmental parameters are likely to be highly correlated, eventual regression models including all variables might be strongly skewed and susceptible to misinterpretation. Therefore, we employed a stepwise selection of variables based on the variance inflation factor (VIF), which is an estimator of multicollinearity among variables (Quinn and Keough, 2002). As a rule of thumb, VIF values greater than 10 indicate the presence of multicollinearity (Quinn and Keough, 2002); some authors even consider values above 5 evidence of collinearity (Heiberger and Holland, 2004). The applied function iteratively removes collinear variables by calculating the VIF of variables against each other (for the script, see Ijaz, 2013); R package “fmsb” v. 0.5.2 (Nakazawa, 2015) is required for this procedure. VIF values were calculated with package “HH” v. 3.1-32 (Heiberger, 2016). To enhance the models further, multiple regressions using backward stepwise selection through an evaluation based on the Akaike information criterion (AIC) were performed with the remaining set of factors. The normality of model residuals was tested with the Shapiro–Wilk tests. In case normality was not achieved, residual distributions were assessed qualitatively using Q–Q plots; only if the majority of cases matched the expected distribution was a model considered significant. Finally, we used the R package “hier.part” v. 1.0-4 (Walsh and Mac Nally, 2013) to evaluate the independent contribution of each predictor to the reduced models.
Relative warp analyses of left
The relative warp analysis (RWA) yielded different results for males and females, while patterns were largely consistent within sexes (Figs. 2, 3). Along the first three relative warps, Mexican females have little overlap with Brazilian and Floridian ones. Only some of the specimens from Punta Laguna in northern Yucatan seem to be morphologically closer to the Floridian group and cluster apart in the analyses of both valves. Brazilian and Floridian specimens have a distinctly higher overlap and differentiate only little along RW 2. A clear differentiation within both clusters, like in the Mexican group, is lacking. Group differentiation in male valves is quite the contrary: Floridian specimens have little overlap with Brazilian ones in both valves along RW 1, while Mexican specimens are hardly separable from either group along any of the first 3 RWs. However, the differentiation between some Punta Laguna valves and the remaining Mexican carapaces along RW 1 is comparable to the patterns observed for females. Mexican and Brazilian males show slight biogeographic differentiation along RW 2 (left valves) and RW 3 (right valves), respectively. No clustering is observed for higher warps in either sex or valve.
First axis of the PLS analysis of carapace shape and environmental variables. Colours refer to the different regions (blue – Florida, green – Mexico, light green – Lake Punta Laguna, pink – Brazil).
Similar to the patterns posed by the scatter plots, the thin-plate splines
indicate that shape variation along the RWs is largely consistent within
valves but differs slightly between sexes. Here we discuss only axes along
which biogeographic discrimination is observed (see Wrozyna et al.,2016, for
inter-group variations). The most important morphological characteristic
representing shape differences along RW 1 in both females and males and right
and left valves is relative carapace length (Figs. 2, 3). However, the exact
expression differs between sexes: valve outline in males varies between
elongated elliptical and short asymmetrical with a slightly inflated anterior
part, and between elongated elliptical and short asymmetrical with distinctly
inflated posterior region (i.e. brood pouch) in females. In addition to
outline differences, the position of the anteriormost pore conulus (LM 2)
shifts in the dorsoventral direction consistently in both valves and sexes.
In females, also the position of the dorsal dip point of the posterior
curvature (LM 6) varies in the dorsoventral direction. The shape variation
along RW 2 is similar for females as in RW 1 but with a different combination
of traits: negative scores correspond to elongated valves with inflated
posterior and slightly shifted LM 2 and LM 6 in the dorsoventral direction.
In male
The PLS analyses indicate relationships between morphological and
environmental variables, yet with different results for males and females.
The first PLS axis explains between 68.7 % and 77.9 % of the total
variation, whereas values are consistently higher for females (LV:
77.5 %; RV: 77.9 %) than for males (LV: 68.7 %; RV: 71.5 %).
In all four analyses, Brazilian specimens are widely separated from Floridian
and Mexican ones along PLS axis 1, corresponding to a clear differentiation
in both environmental and morphological scores. The left valves of females
and the left and right valves of males of Brazilian specimens exhibit
negative scores on both PLS axes corresponding to shape and environmental
variables. Females display inverse distributions for Brazilian and Mexican
specimens. Floridian and Mexican groups have little but consistent overlap in
all analyses, while the specimens of Florida tend to have smaller variation
ranges than Mexican groups (Fig. 4). Permutation tests indicate, however,
that PLS analyses are hardly significant for male valves (LV:
Summary of statistics of the RWA, PLS analyses and multiple regression analyses for RWs 1–3 for the four datasets. The reification column summarizes shape variation along each respective warp. For the multiple regressions, percentage values are indicated for predictor variables with relative contributions over 10 %. BIO2 represents the mean diurnal temperature range, BIO4 represents temperature seasonality, and BIO12 represents annual precipitation. See “Methods” for details.
Continued.
For PLS axis 2, few relationships between shape and environment are yielded for all four datasets, and none of them are significant (see Table S3).
The loadings for morphological variables in the PLS analyses yield constantly high values for RW 1; RW 2 shows loading values higher than the mean (based on absolute values) in all analyses except male right valves; RW 3, in turn, contributes above average to variation in all cases except female right valves. Other warps were not considered because of their minor influence on shape variation (low loading values) or the lack of biogeographic separation. See Table 1 for a summary of the results.
The following warps fulfil the selection criteria defined in ”Methods” for
consideration in the multiple regressions: RW 1 for all four datasets; RW 2
for female right and left valves; RW 3 for female left valves and male right
and left valves. Hence, nine regression analyses were carried out.
Shapiro–Wilk tests of model residuals indicate normality for four of the
nine analyses (Table 1). An inspection of Q–Q plots yielded, however, that
in all models the majority of cases match the expected distributions, which
is why the remaining models are still considered significant (see Supplement
Fig. S1). Eight out of nine models are significant (
Only a limited set of predictor variables is retained out of the original
15 variables in each model after the elimination of collinear parameters and
the backward stepwise selection (see Supplement Table 4). Seven parameters do
not contribute to any models: These are
The link between shape variation and environmental conditions is a
well-studied branch of ostracodology, but studies have yielded quite
contrasting results. Frequently identified ecological factors are the
salinity (Yin et al., 1999, 2001; Grossi et al., 2017) and hydrochemical
regime, mirrored by the
The deduction of general relationships between shape and environmental conditions is hampered due to different approaches, geographical areas and ranges, and different environmental datasets. Many studies are based on mesocosm experiments (e.g. Mezquita et al., 1999; Yin et al., 1999; van Harten, 2000; Frenzel et al., 2012). Although they play an important role in increasing our understanding of the ecophenotypic responses of ostracods to environmental changes, they cannot, by nature, cover the full range and interplay of natural conditions. Other authors studied field populations covering small geographical areas with high resolution (e.g. van der Meeren et al., 2010) or larger study regions with widespread sampling that did not match the distribution of the species involved (e.g. Baltanas et al., 2002; Ramos et al., 2015; Boomer et al., 2017). Our approach is the first that covers a supra-regional scale that coincides with the geographical range of the study taxon. Another novel contribution to the investigation of ecophenotypy is represented by the inclusion of climatic data, in contrast to many other approaches where datasets are often restricted to hydrochemical information. In contrast to climatic data, hydrochemical conditions usually vary on small spatial scales. Previous studies were therefore restricted to characterize local rather than regional effects of the environment on shape changes. It is known from several other organism groups that species may exhibit differences in sensitivity to the ecological conditions in their geographical ranges, since they have a higher sensitivity of range-edge populations than those nearer to the centre of the species' distribution (e.g. Mills et al., 2017). Thus, some environmental parameters that were identified in other studies as major controls on morphological changes (such as salinity) could be important on more restricted geographical scales due to a higher sensitivity of the local populations. The ecophenotypic response to changes of environmental conditions could vary along with the geographical scale. Our approach enables the investigation of the overall pattern of ecophenotypic responses to environmental change and minimizes local effects.
So far, this approach has been the first to study the relationship between
shape and the environment in an ostracod species that occurs in tropical and
subtropical areas. However, little information is available regarding the
biology of
Variation in temperature seasonality, annual precipitation and anions
(
The geographical range of
Temperature has a direct effect on other environmental parameters such as
salinity and the oxygenation of the water. Water temperature is one of the
most important variables affecting metabolism, oxygen consumption, growth,
moulting, and the survival of crustaceans (Le Moullac and Haffner, 2000, and
references therein). The time span of ostracod life cycles varies from a few
months to as long as four years, producing one or more generations per year
(Horne, 2005). At least in temperate regions, the start of the reproductive
period (and thus the moult cycle) is often related to temperature (Van
Doninck et al., 2003). Meyer et al. (2017)
recently found that populations of
Increases in temperature can result in significantly shortened intermoult
periods, higher moulting rates (Roca and Wansard, 1997; Mezquita et al.,
1999; Brylawski and Miller, 2006), increased growth increments (Martens,
1985; Iguchi and Ikeda, 2004), and a reduction in maturation time (Pöckl,
1992). We expect that a stronger temperature seasonality induced prolonged moult cycles in populations of
Precipitation causes declines in nutrients and promotes the physical
disturbance of the water column (Figueredo and Giani, 2009). Moreover,
changes in precipitation directly influence hydrochemical composition and the
input of sediments, organic components, and contaminants as well as affecting
the lake level (Mortsch and Quinn, 1996; Whitehead et al., 2009). This also
includes indirect influences, e.g. those affecting aquatic plants, which
represent important microhabitats and/or food sources (Lacoul and Freedman,
2006). The annual cycle of precipitation over most of South America is
monsoon-like, with great contrasts between winter and summer (Grimm et al.,
2007). The peak rainy season in the Brazilian sample region is during the
austral winter. The rainfall is caused by frontal penetration associated with
migratory extratropical cyclones (Grimm et al., 1998). The amount of rainfall
in Yucatan is associated with the seasonal migration of the Intertropical
Convergence Zone and to a lesser extent with spatially oriented tropical
convective activity (e.g. Hodell et al., 2008). Florida, in particular
southern Florida where most of our samples are derived from, receives maximum
precipitation during the summer in the Northern Hemisphere due to
convectional and tropical storms (Schmidt et al., 2001). The annual
precipitation amounts for the sampled areas are higher on average in Brazil
at 1396–1492 mm per year compared to Florida and Yucatan, with 1185–1430
and 1125–1359 mm, respectively. Since the annual precipitation amounts of
the regions are very similar, it might be more plausible that precipitation
seasonality has an influence on the carapace shape of
The ionic composition of the host water is vital for the calcification and
growth rates of ostracods (Mezquita et al., 1999). The relationship between
hydrochemistry and phenotypic variability is poorly understood, however. A
study from Kim et al. (2015) shows that increased levels of pH account for
decreased carapace growth rates, i.e. prolonged intermoult periods, and
smaller carapaces. Carapace shape differences have been moreover associated
with changes in
Natural sources of
Van der Meeren et al. (2010) found ostracod valve shape variability to
significantly correlate with the ratio between alkalinity and sulfate. As the
ratio inversely related to solute concentration, the authors hypothesized
that carapace shape may be linked to changes in the lake water balance,
relative climatic moisture, or changes in the sources of solutes delivered to
the environment. Varying anionic composition has also been considered to
affect osmoregulation and calcification (Mezquita et al., 1999). As
hyperosmotic organisms, freshwater ostracods pump ions inwards (mainly
One of the best-studied phenomena in ostracods is variable noding (hollow
outward flexions on the lateral surfaces on the valves) in
Phenotypic variation in ostracods is considered to reflect either genotypic
or ecophenotypic variability or a combination of both (Martens et al., 1998;
Yin et al., 1999; Anadón et al., 2002; Frenzel and Boomer, 2005; Boomer
et al., 2017; Grossi et al., 2017). A recent study on the valve outline
variability of a non-marine ostracod demonstrated that differences in
carapace shape do not correspond to genetic clades (Koenders et al., 2016).
However, caution is advised when comparing patterns among species, since
different species react differently and have varying potentials for
ecophenotypic variation (Anadón et al., 2002; Frenzel and Boomer, 2005).
The relationship between genotype and environment might differ among species,
geographical regions, and over time (see, e.g. Sanchez-Gonzalez et al., 2004;
Koenders et al., 2016). Our results clearly imply that morphological
disparity in
The comparison of our results with a large number of previous studies speaks
to the difficile nature of the ecophenotypic response to varying climatic and
ecological conditions in freshwater ostracods. Shape variation in
Temperature (per se), salinity (expressed as electrical conductivity), and pH
have had surprisingly little or no effects on shape variation in
All data are presented within the paper or in the Supplement. Bioclimatic data are provided by the WorldClim database (WorldClim, 2017).
CW, JM, and WEP carried out sampling of
The authors declare that they have no conflict of interest.
We are grateful to Norma Luíza Würdig (Universidade Federal do Rio Grande do Sul and personnel at CECLIMAR in Tramandai, Brazil) for offering their facilities. We thank Øyvind Hammer (University of Oslo) for helping with the permutation tests. We also want to thank Thomas Wagner (University Graz) for his helpful comments in the hydrological and hydrogeological discussion. Financial support was provided by the Austrian Science Fund (grant number P26554). Thomas A. Neubauer was supported by a Just'us postdoctoral fellowship granted by the University of Giessen and an Alexander-von-Humboldt Fellowship for postdoctoral researchers. We thank three anonymous reviewers for their helpful comments. Edited by: Nobuhito Ohte Reviewed by: three anonymous referees