Supplemental Information for:

Rapid colour shift by reproductive character displacement in Cupido butterflies

Joan Carles Hinojosa, Darina Koubínová, Vlad Dincă, Juan Hernández-Roldán, Miguel L.

Munguira, Enrique García-Barros, Marta Vila, Nadir Alvarez, Marko Mutanen & Roger Vila

Notes on morphology

Prieto et al. (2009) found differences in wing patterns between the taxon carswelli and C.

lorquinii using multivariate morphometrics, while they stated that genital structures of C.

minimus and carswelli are indistinguishable. On the other hand, subtle differences in genitalia

between C. lorquinii, C. minimus and carswelli have been stated by Gil-T (2006). In contrast, in

our material C. lorquinii genitalia (Figure S2) strongly resemble those of carswelli as described

by Gil-T (2006). No morphological differences have been described between the larvae of

carswelli and C. lorquinii, and the only difference observed in the preimaginal stages is the

yellow pilosity of the pupa in carswelli, compared to white in C. lorquinii (Gil-T, 2006).

Notes on ecology

Cupido lorquinii and carswelli are regularly found on calcareous substrates (García-Barros et al.,

2013; Gil-T, 2003) and larvae feed on the same host plant, Anthyllis vulneraria (Fabaceae).

Slightly distinct ecological preferences were mentioned in Gil-T (2017), the most important of

which is that carswelli populations occur at 1,000-1,800 m.a.s.l. (Tolman & Lewington, 2008),

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whereas C. lorquinii has a wider range of 0-2,600 m.a.s.l. (Gil-T, 2017; Tennent, 1993). Despite

this, we observed both taxa in the same type of habitats: at altitudes of about 1,000 m (and

lower for C. lorquinii) in relatively humid areas with low scrub cover in forest clearings and at

higher altitudes in rocky environments, including torrents.

Notes about introgression in the subgenus Cupido

We found evidence that all the European species of the subgenus Cupido can occasionally

interbreed successfully, or at least introgression events seem to have occurred in the past. The

mitochondrial phylogeny (Figure 2) suggests at least one introgression event from C. minimus to

C. osiris (a male of the latter species from Granada, southern Iberia, clustered with C. minimus

in the COI phylogeny), although incomplete lineage sorting cannot be discarded. Two strains of

Wolbachia were shared across species (Table S3): one strain was present in some specimens of

C. minimus and C. lorquinii, and another was present in C. osiris and in one C. minimus. These

results apparently support the idea that C. lorquinii introgressed C. minimus and C. minimus

introgressed C. osiris. However, the ABBA-BABA test did not detected signal of introgression

between C. minimus and C. lorquinii, and instead a weak signal, not significant, was obtained for

introgression between C. minimus and carswelli (p-value=0.3-0.42, D=0.0067, fG=0.0041).

Cupido minimus male colour is brown, commonly with a few blue scales. In some parts of its

distribution where C. osiris is absent, such as in the UK or northern Europe, blue scales in males

can be sometimes abundant. Given that wrong courtships between C. minimus and C. osiris

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may take place, reduction in the number of blue scales in C. minimus may be related to the

presence of C. osiris and could have resulted from a reproductive character displacement

process similar to that we describe in carswelli.

REFERENCES

García-Barros, E., Munguira, M. L., Stefanescu, C., Vives Moreno, A. (2013). Fauna IbéricaVolumen 37: Lepidoptera: Papilionoidea. Madrid, Spain: Consejo Superior de InvestigacionesCientíficas.

Gil-T, F. (2003). Cupido carswelli (Stempffer, 1927): descripción de sus estadios preimaginales,biología y distribución. La morfología de la crisálida, ¿Clave para su rango específico?(Lepidoptera, Lycaenidae). Boletín de la SEA, 32, 45–50.

Gil-T, F. (2006). Cupido carswelli (Stempffer, 1927): morphology of its chrysalis and genitaliacompared with those of Cupido minimus (Fuessly, 1775) and Cupido lorquinii (Herrich-Schäffer, 1847) (Lepidoptera, Lycaenidae). Atalanta, 37, 150–160.

Gil-T, F. (2017). Cupido carswelli (Stempffer, 1927), endemism from SE. Spain, a differentspecies of Cupido minimus (Fuessly, 1775): updated distribution, identification of its larvalhost-plant and notes on taxonomy, ecology and morphology (Lepidoptera, Lycaenidae).Atalanta, 48, 197–203.

Tennent, J. (1993). The butterflies of Morocco, Algeria and Tunisia. Swindon, UK: Swindon PressLimited.

Tolman, T., & Lewington, R. (2008). Collins Butterfly Guide. London, UK: Harper-CollinsPublishers.

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Supplementary tables

Table S1. Samples used in this study. The first missing data column is the percentage of missing

data data after IPYRAD pipeline in the dataset with all the samples and the second column is

the same value but in the dataset with only C. l. carswelli and C. l. lorquinii.

Sample ID Species Genbank code (COI) Sex Wingcolour

Locality Country Missing,dataset 1

(%)

Missing,dataset 2

(%)

RVcoll09V159 C. l. carswelli KP870815 Male Brown Sierra María Spain 41.1 29.7

RVcoll09V160 C. l. carswelli KP870825 Male Brown Sierra María Spain 39.2 28.2

RVcoll09V161 C. l. carswelli KP870888 Male Brown Sierra de Orce Spain 46.6 35.7

RVcoll15R285 C. l. carswelli MT763230 Male Brown Sierra de Guillimona Spain 87.8 84.6

RVcoll17B236 C. l. carswelli MT763233 Male Brown Sierra Espuña Spain 46.6 36.7

RVcoll17B238 C. l. carswelli MT763205 Male Brown Sierra Espuña Spain 43.7 33.1

RVcoll17B326 C. l. carswelli MT763231 Male Brown Sierra de las Cabras Spain 40.6 29.7

RVcoll17B327 C. l. carswelli MT763219 Male Brown Sierra de las Cabras Spain 49.9 39.6

RVcoll17B665 C. l. carswelli MT763224 Male Brown Puebla de Don Fadrique Spain 39.7 28.6

RVcoll17B741 C. l. carswelli MT763221 Female Brown Sierra de Segura Spain 56.5 47.7

RVcoll06G608 C. l. lorquinii MT763218 Male Blue Col du Zad Morocco 62.8 54.3

RVcoll08H623 C. l. lorquinii HM901583 Male Blue Benadalid Spain 53.4 43.4

RVcoll08J206 C. l. lorquinii HM901592 Male Blue Sierra Almijara Spain 76.8 71.4

RVcoll08L023 C. l. lorquinii GU676451 Male Blue Casillas de Rojas, Güejar Sierra Spain 52.2 41.8

RVcoll08L352 C. l. lorquinii GU676356 Male Blue Sierra Prieta Spain 57.4 48.0

RVcoll08L674 C. l. lorquinii HM901621 Female Brown Iznalloz, Sierra Arana Spain 55.1 45.4

RVcoll11D612 C. l. lorquinii KP870431 Male Blue Cañada Real de la Zubia, Monachil Spain 41.7 30.1

RVcoll13S440 C. l. lorquinii MT763212 Male Blue Aït-Lekak, NE Oukaïmeden Morocco 80.4 75.8

RVcoll13S441 C. l. lorquinii MT763222 Male Blue Adrar-n-Aklim, E Igherm Morocco 74.3 68.1

RVcoll16A189 C. l. lorquinii MT763210 Male Blue Inifife, South Timahdite, Moyen Atlas Morocco 75.7 69.9

RVcoll17B433 C. l. lorquinii MT763214 Male Brown Sierra de Huétor Spain 43.0 32.3

RVcoll17B434 C. l. lorquinii MT763209 Female Brown Sierra de Huétor Spain 50.7 40.2

RVcoll17B440 C. l. lorquinii MT763207 Male Blue Sierra de Huétor Spain 64.6 57.3

RVcoll17B441 C. l. lorquinii MT763228 Male Blue Alcaucín Spain 60.5 54.8

RVcoll17B442 C. l. lorquinii MT763220 Male Blue Sierra de Huétor Spain 62.8 54.6

RVcoll17B498 C. l. lorquinii MT763216 Male Blue Zuheros Spain 51.0 40.2

RVcoll17B636 C. l. lorquinii MT763217 Female Brown Sierra de los Filabres Spain 62.3 54.1

RVcoll17B806 C. l. lorquinii MT763229 Male Blue Grazalema Spain 54.1 44.0

RVcoll17B859 C. l. lorquinii MT763223 Male Blue Monchique Portugal 53.0 42.7

RVcoll17B860 C. l. lorquinii MT763211 Male Blue Monchique Portugal 62.6 54.4

RVcoll06N004 C. minimus HQ004334 - Brown Rimetea Romania 63.2 -

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RVcoll08M911 C. minimus GU669864 - Brown Bassa d'Arrès, Val d'Aran Spain 51.8 -

RVcoll11D667 C. minimus KP871105 - Brown Font de l'Arbre, Serra d'Aitana Spain 57.9 -

RVcoll11G699 C. minimus MT763204 - Brown Uspenka Kazakhstan 58.0 -

RVcoll12M489 C. minimus MN143183 - Brown Monte Limina, Aspromonte Italy 57.4 -

RVcoll12M574 C. minimus MT763226 - Brown Nebrodi, Floresta Italy 52.4 -

RVcoll12Z144 C. minimus MN141369 - Brown Gortlecka (the Burren) Ireland 52.2 -

RVcoll14H750 C. minimus MT763206 - Brown Peristera Greece 51.5 -

RVcoll14I802 C. minimus MT763232 - Brown Tatranska Kotlina, Tatra Mt. Slovakia 49.7 -

RVcoll15H695 C. minimus MT763208 - Brown Splugenpass Switzerland 72.3 -

RVcoll16G496 C. minimus MT763225 - Brown Rå Sweden 70.0 -

RVcoll17A293 C. minimus MT763213 - Brown Quatretonda Spain 50.2 -

RVcoll17A892 C. minimus MT763215 - Brown Kovacic Croatia 57.4 -

RVcoll08M939 C. osiris HM901312 - - Sobrecastells Spain 75.2 -

RVcoll17B693 C. osiris MT763227 - - Puebla de Don Fadrique Spain 71.5 -

Table S2. Number of loci and SNPs of each dataset and analyses where they have been used.

IPYRAD raw output IPYRAD raw output (unlinked) CENTRIFUGE output Rare allele filtering output

All samples 17,825 loci; 130,679 SNPs 16,562 SNPs 16,312 loci; 109,221 SNPs 62,533 SNPs

Usage Coancestry matrix STRUCTURE (supplementrary) ML phylogeny, D-statistics PCA, STRUCTURE

Only C. l. lorquinii and C. l. carswelli 17,522 loci; 81,803 SNPs 15,333 SNPs 16,030 loci; 69,327 SNPs 45,753 SNPs

Usage BAYESCAN - - PCA, STRUCTURE

Table S3. Minimum COI (658 bp) genetic distances (dXY) between the studied taxa.

C. l. lorquinii C. l. carswelli C. minimus C. osiris

C. l. lorquinii -

C. l. carswelli 0% -

C. minimus 1.3% 1.3% -

C. osiris 2% 2% 0.6% -

Table S4. Top hits retrieved in BLAST Lepbase. The target sequence was the sole BAYESCAN

outlier that had exclusive haplotypes for all the brown males, including C. l. carswelli and the

brown C. l. lorquinii.

subject id Identity

(%) alignment

length mismatches

gapopens

q.start

q.end

s. start s. end E value bit

score

calycopis_cecrops_v1x1_core_32_85_1__scaffold__scaffold37 74,41 211 29 2 1 186 139367 139577 4E-34 152

operophtera_brumata_v1_core_32_85_1__scaffold__OBRU01_Sc00484 83,21 137 15 2 58 186 77627 77491 8E-31 141

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calycopis_cecrops_v1x1_core_32_85_1__cds__cce37.3.mRNA 78,62 145 20 1 51 184 532 388 2E-26 127

phoebis_sennae_v1x1_core_32_85_1__scaffold__scaffold141_cov78 79,73 148 25 3 44 186 16481 16334 2E-25 123

bicyclus_anynana_v1x2_core_32_85_1__scaffold__BANY00291 79,71 138 23 2 54 186 207138 207001 9E-24 118

bicyclus_anynana_nba01_core_32_85_1__scaffold__nBa.0.1.scaf01317 79,71 138 23 2 54 186 66419 66556 9E-24 118

bicyclus_anynana_v1x2_core_32_85_1__cds__BANY.1.2.t15379 79,41 136 23 2 54 184 949 814 1E-22 114

000474F 79,41 136 26 2 53 186 162085 161950 4E-22 113

000448F 79,41 136 26 2 53 186 24755 24890 4E-22 113

junonia_coenia_jc10_core_32_85_1__cds__JC_0017693-RA 79,1 134 26 2 53 184 443 310 5E-21 109

Table S5. Genetic distances (dXY) of the concatenated Wolbachia loci (2,435 bp). Invariant loci

and individuals with only one or two loci were removed. Strain numbers are written in brackets.

C. osirisRVcoll17B693

Spain (S2)

C. minimusRVcoll11D667

Spain (S1)

C. osirisRVcoll08M939

Spain (S2)

C. l. lorquiniiRVcoll17B441

Spain (S1)

C. l. lorquiniiRVcoll17B860Portugal (S3)

C. minimusRVcoll16G496Sweden (S2)

C. minimusRVcoll08M911

Spain (S1)

C. osirisRvcoll17B693

Spain

C. minimusRVcoll11D667

Spain (S1)2.16%

C. osirisRVcoll08M939

Spain (S2)0.00% 2.44%

C. l. lorquiniiRVcoll17B441

Spain (S1)2.16% 0.00% 2.44%

C. l. lorquiniiRVcoll17B860Portugal (S3)

2.12% 1.85% 1.95% 1.85%

C. minimusRVcoll16G496Sweden (S2)

0.00% 1.69% 0.00% 1.69% 1.82%

C. minimusRVcoll08M911

Spain (S1)1.39% 0.00% 1.34% 0.00% 1.82% 0.97%

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Supplementary figures

Figure S1. Distribution maps of all the taxa included in the study with the collecting sites

indicated by black dots.

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Figure S2. Wing and genitalia pictures of the C. lorquinii males caught in Sierra de Huétor

(Granada, Spain). The external morphology of specimen RVcoll17B433 is virtually identical to

the taxon carswelli, with brown upperside wings that have very few and scattered blues scales.

The scale is the same in each row. Black bars indicate 0.5 mm (not applicable for the wings).

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Figure S3. Coancestry matrix and phylogeny retrieved by fineRADstructure (17,825 loci). Black

boxes correspond to C. minimus, grey to C. l. lorquinii and reddish to C. l. carswelli.

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Figure S4. STRUCTURE results (K = 3) based on 16,562 SNPs (unlinked) and 62,533 SNPs (also

used in Figure 3).

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Figure S5. STRUCTURE results (K = 2, K = 3) based on the dataset including only C. l. lorquinii and

C. l. carswelli (45,753 SNPs).

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