Genetics redraws pelagic biogeography of Calanus

Planktonic copepods of the genus Calanus play a central role in North Atlantic/Arctic marine food webs. Here, using molecular markers, we redrew the distributional ranges of Calanus species inhabiting the North Atlantic and Arctic Oceans and revealed much wider and more broadly overlapping distributions than previously described. The Arctic shelf species, C. glacialis, dominated the zooplankton assemblage of many Norwegian fjords, where only C. finmarchicus has been reported previously. In these fjords, high occurrences of the Arctic species C. hyperboreus were also found. Molecular markers revealed that the most common method of species identification, prosome length, cannot reliably discriminate the species in Norwegian fjords. Differences in degree of genetic differentiation among fjord populations of the two species suggested that C. glacialis is a more permanent resident of the fjords than C. finmarchicus. We found no evidence of hybridization between the species. Our results indicate a critical need for the wider use of molecular markers to reliably identify and discriminate these morphologically similar copepod species, which serve as important indicators of climate responses.


SUPPLEMENTARY 1 Sampling information and molecular-based
Calanus species composition within samples. N individuals per sample were genotyped with nuclear InDel markers. Samples relative proportions of C. glacialis (Cgla), C. finmarchicus (Cfin), C. hyperboreus (Chyp) and C. helgolandicus (Chel) are reported in percentage. In the Laptev Sea and the Nansen Basin, only species presence (x) or absence (-) is reported.

Frequency distributions of prosome length for Calanus glacialis and C. finmarchicus at developmental stage (a) CV and (b) adult female in the region of Saltenfjord / Skjerstadfjord
In total, prosome length of 171 Calanus individuals was measured. of InDels. Each individual is represented by a bar filled with one or two distinct colours that identify an individual probability to belong to two clusters (here, green for C. finmarchicus and red for C. glacialis). In case of F1 hybrids between C. finmarchicus and C. glacialis, a bar will be nearly equally filled with both colours (which never happened).

SUPPLEMENTARY 7 Supplementary Protocols
v DNA extraction: We extracted DNA from the antennules of each specimen, using the quick and cheap method of HotSHOT DNA extraction [2]: 1-Individuals were soaked separately in sterile water to rinse the ethanol; 2-One by one, under a stereomicroscope, the 2 antennules were removed from the rest of the body and placed in 50 µL of a Lysis Buffer (See HotSHOT protocol for details about composition of buffers [2]) in a 96-well plate; 3-The plate was incubed in a thermocycler, 30 minutes at 95°C; 4-The plate was subsequently cooled in the fridge (4°C) for 5-10 minutes; 5-Finally, 50 µL of Neutralizing Solution was added (See HotSHOT protocol for details about composition of buffers [2]).
v Molecular species identification: We amplified a set of 6 nuclear molecular markers, type InDel (polymorphism consists of Insertion or Deletion of nucleotides): G_150, T_461, T_1338, T_1966, T_3133 and T_4700 [3] in a single multiplexed Polymerase Chain Reaction (PCR), and genotyped them following the protocol described by Smolina et al. (2014) [3]. Four distinct patterns of genotypes were distinguished and assigned to the four different species of Calanus based on species-specific alleles defined in Smolina et al. (2014) [3]. This method is fast and inexpensive. A total of 96 individuals can be reliably identified within 5 hours, with 100% reliable results for ca. 2 euros/sample. At one point in our study, we had to re-order a new stock of InDel primers from a new provider, and thus had to change the type of fluorescent dye labelling of the forward primers (from 6-FAM, VIC and NED (Life Technologies) to FAM, YAKYE and ATTO550 (Eurofins Genomics)). This resulted in a slight shift of the length of the alleles in the genotyping, thus this second set of data was treated separately. To confirm the species identification, and in order to validate our nuclear markers, we sequenced a portion of the mitochondrial 16S rDNA (ca. 360bp) [4] for 159 individuals from 53 locations selected to represent the full range of sampling, and for 129 individuals from the region of Saltenfjord / Skjerstadfjord, following the same protocol described in Smolina et al. (2014) [3]. The obtained sequences were then aligned together with one reference 16S sequence for each species from GenBank ® : HQ266740 for C. glacialis, AF295334 for C. finmarchicus, KF956849 for C. helgolandicus, and JX678968 for C. hyperboreus. This alignment was used to reconstruct a PhyML tree (GTR model) using Geneious version 9.1 (http://www.geneious.com) [5]. The resulting tree displayed four clearly distinct groups of sequences corresponding to the four species (see Supplementary2). In all individuals, this approach resulted in the same species identification as the InDel genotyping (Supplementary3-4).
v Microsatellite analysis: To characterize connectivity among newly described population of C. glacialis in Norwegian fjords and other regions and compare it to C. finmarchicus we performed analysis of population genetic differentiation using sample from 3 locations: Isfjord, Saltfjord and Lurefjord (c.f. Supplementary1). DNA from the antennas of 24 identified (InDels method -see above) individuals per species and per location was used to amplify 10 microsatellites markers [6,7] [10].

Sources of literature used for tracing the morphologically based distribution ranges of Calanus species
The map showing the distribution ranges of Calanus species in the North Atlantic and Arctic Ocean, as defined from morphological identification of species, presented as Figure 1 of the paper, was mainly based on three different sources : Conover, 1988[11], Barnard et al., 2004[12] for the southern borders of species distributions, and Jaschnov, 1970 [13]