# Morphometric and Molecular Discrimination of the sugarcane aphid, Melanaphis sacchari (Zehntner, 1897) and the sorghum aphid Melanaphis sorghi (Theobald, 1904)

Samuel Nibouche, Laurent Costet, Raul F Medina, Jocelyn R Holt, Joëlle Sadeyen, Anne-Sophie Zoogones, Paul Brown, Roger L Blackman

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## To cite this version:

Samuel Nibouche, Laurent Costet, Raul F Medina, Jocelyn R Holt, Joëlle Sadeyen, et al.. Morphometric and Molecular Discrimination of the sugarcane aphid, Melanaphis sacchari (Zehntner, 1897) and the sorghum aphid Melanaphis sorghi (Theobald, 1904). PLoS ONE, 2020, 16 (3), pp.e0241881. 10.1101/2020.10.23.351833. [hal-03047852](https://hal-univ-reunion.fr/hal-03047852)

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1 Morphometric and Molecular

2 Discrimination of the sugarcane aphid,

3 *Melanaphis sacchari*, (Zehntner, 1897)

4 and the sorghum aphid *Melanaphis*

5 *sorghi* (Theobald, 1904)

6 Samuel Nibouche¹*, Laurent Costet¹, Raul F. Medina², Jocelyn R. Holt², Joëlle Sadeyen³,

7 Anne-Sophie Zoogones¹,³, Paul Brown⁴, Roger L. Blackman⁴

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10 ¹CIRAD, UMR PVBMT, F-97410 Saint Pierre, La Réunion, France

11 ²Texas A&M University, College Station, Texas, United States of America

12 ³Université de La Réunion, UMR PVBMT, F-97410 Saint Pierre, La Réunion, France

13 ⁴The Natural History Museum, London SW7 5BD, United Kingdom

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15 * Corresponding author

16 A-mail: samuel.nibouche@cirad.fr

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# Abstract

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*Melanaphis sacchari* (Zehntner, 1897) and *Melanaphis sorghi* (Theobald, 1904) are major worldwide crop pests causing direct feeding damage on sorghum and transmitting viruses to sugarcane. It is common in the scientific literature to consider these two species as synonyms, referred to as the ‘sugarcane aphid’, although no formal study has validated this synonymy. In this study, based on the comparison of samples collected from their whole distribution area, we use both morphometric and molecular data to better characterize the discrimination between *M. sacchari* and *M. sorghi*. An unsupervised multivariate analysis of morphometric data clearly confirmed the separation of the two species. The best discriminating characters separating these species were length of the antenna processus terminalis relative to length of hind tibia, siphunculus or cauda. However, those criteria sometimes do not allow an unambiguous identification. Bayesian clustering based on microsatellite data delimited two clusters, which corresponded to the morphological species separation. The DNA sequencing of three nuclear and three mitochondrial regions revealed slight divergence between species. In particular, the COI barcode region proved to be uninformative for species separation because one haplotype is shared by both species. In contrast, one SNP located on the nuclear EF1-α gene was diagnostic for species separation. Based on morphological and molecular evidence, the invasive genotype damaging to sorghum in the US, Mexico and the Caribbean since 2013 is found to be *M. sorghi*.

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# Introduction

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The species *Melanaphis sacchari* (Zehntner, 1897) and *Melanaphis sorghi* (Theobald, 1904) were described at the turn of the 20th century by Zehntner [1] on sugarcane in Java and by Theobald [2] on sorghum in Sudan respectively. However, although these two species are commonly treated as synonyms, referred to as the ‘sugarcane aphid’, no comparative study demonstrating this synonymy has been conducted. When Remaudière & Remaudière [3] considered *M. sorghi* as a synonym of *M. sacchari* in their 1997 catalogue, following Eastop (1953) [4], they provided no reference to support this choice. Moreover, Halbert and
Remaudière [5] referred later to these species as the 'sorghi/sacchari group' and underlined them as 'two very variable species usually regarded as synonyms, but possibly distinct according to Blackman et al. (1990)'. The communication by Blackman et al. [6] indeed supported the separation of *M. sorghi* and *M. sacchari* and provided a morphological criterion to separate the species, based on the ratio between the hind tibia length and the antennal processus terminalis length. The same separation was used in Blackman and Eastop's book in 2006 [7] and has not been challenged since.

Margaritopoulos et al. [8] stated that 'DNA evidence that might confirm the existence of two species is not yet available, but at this time it seems advisable to recognise that they are probably functioning as distinct taxonomic entities'. Nibouche et al. [9] observed genetic structuring in five clonal lineages matching a geographic structure, but they could not separate the two species by using 'universal' COI barcoding.

Regarding host plant association, each species is observed on both sugarcane and sorghum, but *M. sorghi* is considered preferring sorghum and *M. sacchari* preferring sugarcane [6]. However, host plant association is blurred by the existence of biotypes, as shown by Nibouche et al. [10] who demonstrated the existence of a sorghum and a sugarcane biotype in Reunion populations (within the same multi locus lineage). Interestingly, *Melanaphis sorghi* is known for a long time to produce very heavy infestations on sorghum, in Africa. Early in the 20th century, Vuillet & Vuillet (1914) [11] cited this aphid as responsible for famines in West Africa.

The objectives of this study were (1) to improve the description of the morphometric differences between *M. sorghi* and *M. sacchari*, and (2) to delimit the molecular separation of *M. sorghi* and *M. sacchari*.
## 70 Material and Methods

### 71 Material collected

This study is based on 199 samples, collected from 2002 to 2016 in 31 states or countries (S1 Table). No specific permissions were required when sampling aphids in the locations studied. From these 199 samples, we analyzed 2,409 apterous aphid specimens collected on cultivated sorghum (Sorghum bicolor) (n = 439), Johnson grass (Sorghum halepense) (n = 97), sugarcane (Saccharum officinarum x S. spontaneum) (n = 1,382), Sorghum arundinaceum (≡ Sorghum bicolor verticiliflorum ≡ Sorghum verticiliflorum) (n = 427), maize (Zea mays) (n = 2), perennial sorghum (Sorghum × almum) (n = 8), Sorghum sp. (n = 16), and pearl millet (Pennisetum glaucum) (n = 38). Most of this material was already analyzed in previous studies [9,10,12]. Using Blackman & Eastop's (2006) key, we identified these specimens as *M. sacchari* or *M. sorghi*, but the identification sometimes was ambiguous given the continuous and overlapping nature of the criteria used to separate both species. For convenience, while awaiting this taxonomy study, this material was referred to as *M. sacchari* in our previous papers.

### 85 DNA extraction

DNA of individual aphids was extracted using the 'salting-out' protocol of Sunnucks and Hales [13] or using the Qiagen DNeasy Blood & Tissue Kit (Qiagen, Courtaboeuf, France). For slide-fixed specimens, a non-destructive DNA extraction was performed using the Qiagen manufacturer's protocol, but retrieving the insect body from the first elution column [12].

### 90 Microsatellites

Nine microsatellite (Single Sequence Repeat, SSR) markers were selected among 14 previously developed markers [14]. PCR reactions were performed with labelled primers and multiplexed following previously established protocols [9]. Genotyping was carried out using
an ABI PRISM 3110 and alleles were identified at each locus by comparison with a size standard using Gene-Mapper version 2.5 software (Applied Biosystems). The total microsatellite genotyping dataset included 2,255 specimens: 2,175 specimens were previously analyzed in [9,10,12] and 80 additional specimens were genotyped for this study. Single combinations of alleles were characterized and arranged as distinct multilocus genotypes (MLG) and assigned to one of six multilocus lineages (MLL) [9,12]. We carried out a Bayesian clustering analysis with Structure version 2.3.4 [15], results were summarized with Structure Harvester [16], Clumpp [17] and Distruct 1.1 [18]. Parameters of the Structure analysis were: admixture, independent allele frequencies, 100,000 iterations after a 25,000 burn-in period, 10 replications for each k value ranging from 1 to 8.

## 104 DNA sequencing

Aphids were sequenced for three mitochondrial and three nuclear DNA regions belonging to the cytochrome c oxidase subunit I (COI) [19], cytochrome c oxidase II (COII) [20], cytochrome b (CytB) [21], elongation factor-1α (EF1-α) [22] and the internal transcribed spacer 1 and 2 (ITS1 and ITS2) [23].

Three of the six sequences, COI, COII and EF1-α, produced informative polymorphism and were simultaneously sequenced on a large number of specimens. These sequences were concatenated and the resulting concatenated haplotypes were used to produce a minimum spanning network with PopArt [24].

## 113 Morphometry

### 114 Material

The morphometry dataset consisted of 89 apterous female specimens that were slide-fixed (S1 Fig) after a non-destructive DNA extraction. Among these 89 specimens, 21 were successfully genotyped with SSR and could be assigned to a multilocus lineage (MLL). The remaining 68 were not genotyped, but belonged to a sample whose multilocus lineage (MLL)
119 had been identified from other specimens. Since 186 of the 188 (98.9%) SSR genotyped
120 samples in this study were homogeneous (i.e. contained only a single MLL), we assumed that
121 the non-genotyped slide-fixed samples belonged to the same MLL as the other specimens in
122 the sample that were genotyped. The distribution of the slide-fixed specimens was: 22 MLL-A,
123 2 MLL-B, 19 MLL-C, 15 MLL-D and 31 MLL-F. Since only two specimens of MLL-B were
124 observed, they were discarded from the statistical analysis. No MLL-E specimen was
125 observed.

126 We also examined the Theobald type series of *Aphis sacchari* collected in Sudan in
127 1902, which is stored in the Natural History Museum of London (NHM). In this series, only one
128 paratype specimen (NHM-1915-81) was an apterous female and could be used for our
129 morphometric analysis. Unfortunately, we were unable to locate the types of *Melanaphis
130 sacchari*. Hollier & Hollier [25] reported that a fire destroyed the experimental station of Salatiga
131 in Java in 1902, including Zehntner's laboratory and his collections. As Zehntner did not send
132 his types to the Geneva museum, the type of *Melanaphis sacchari*, collected in 1897, probably
133 was destroyed and should be considered lost.

134 We also examined some alate *M. sorghi* (n = 5) and *M. sacchari* (n = 5) specimens on
135 slides from the MNHN (Muséum National d'Histoire Naturelle, Paris; G. Remaudière collection)
136 and from the GBGP (Centre de Biologie et de Gestion des Populations, Montpellier; F. Leclant
137 collection). The Theobald's type series from NHM contained one alate specimen (NHM-1915-
138 81), but it was of insufficient quality to be included in the morphometric characterization of
139 alatae.

140 Methods

141 Twenty-two characters that are used classically in aphid taxonomy [7,26] were
142 measured on each slide-fixed specimen, using a binocular lens stereo microscope. Paired
143 appendages (i.e. legs, antenna, siphunculi), were measured on both sides, and the mean value
144 was used for analysis (except for the number of setae on the antenna, which was observed on
145 one side only). Twelve ratios were computed from these characters.
The analysis was carried out on a subset of 11 characters (Table 1) that are known to be discriminant between species within the *Melanaphis* genus [5,7,26,27]. Because of missing data, only 50 specimens were used in the multivariate analysis. Because Theobald did not clarify his *M. sorghi* paratype specimen (NHM-1915-81), we were unable to observe *urs* and *siphBW*, causing three missing ratios in the dataset. To include the paratype in the analysis, we replaced these three missing data by zeros after the standardization step (see below).

<table><caption>**Table 1. List of the 11 morphological variables used in the discriminant analysis of principal components (DAPC) analysis.**</caption><thead><tr><td>Variable name</td><td>Variable signification</td></tr></thead><tbody><tr><td>NsetaeCauda</td><td>number of setae on the cauda</td></tr><tr><td>pt:cauda</td><td>ratio processus terminalis length / cauda length</td></tr><tr><td>HindTibia:pt</td><td>ratio hind tibia length / processus terminalis length</td></tr><tr><td>Ant:BL</td><td>ratio antenna length / body length</td></tr><tr><td>urs:htII</td><td>ratio ultimate rostral segment length / hind tarsa II length</td></tr><tr><td>pt:Vlb</td><td>ratio processus terminalis length / base length of the 6th antennal segment</td></tr><tr><td>pt:siph</td><td>ratio processus terminalis length / siphunculi length</td></tr><tr><td>cauda:urs</td><td>ratio cauda length / ultimate rostral segment length</td></tr><tr><td>siph:BL</td><td>ratio siphunculi length / body length</td></tr><tr><td>siph:siphBW</td><td>ratio siphunculi length / siphunculi basal width</td></tr><tr><td>siph:cauda</td><td>ratio siphunculi length / cauda length</td></tr></tbody></table>

The 11 variables were standardized to obtain a mean of zero and a standard deviation of 1 prior to analysis.

The data were first submitted to a discriminant analysis of principal components (DAPC) with the R package ADEGENET using the *find.clusters* function [28]. This method first uses an unsupervised k-means clustering approach to determine the number of clusters without requiring any a priori clustering information. The determination of the number of
clusters was based on the Bayesian Information Criteria (BIC). Then in a second step, a principal component analysis (PCA) is carried out, followed by a canonical discriminant analysis (CDA) performed on the coordinates along the principal components. To verify if the clustering was congruent with the separation of *M. sorghi* and *M. sacchari*, we compared the *HindTibia:pt* ratio between the clusters delimited by ADEGENET. Indeed, *M. sorghi* specimens have a relative length of the processus terminalis shorter than *M. sacchari* specimens [6,7]. According to [7,8,26], the *HindTibia:pt* ratio range for apterae is (2.0 - 3.0) for *M. sorghi* vs. (1.4 - 2.2) for *M. sacchari*.

Secondly, the complete morphological dataset (i.e. 34 morphological traits on 88 apterous specimens) was submitted to a one-way ANOVA with SAS PROC GLM [29] to detect significant differences between *M. sacchari* and *M. sorghi*. Because we carried out multiple analysis, to control the study-wise type-1 error level we used a 5% False Discovery Rate (FDR) approach [30] with SAS PROC MULTTEST [29] to detect significant differences between clusters. To carry out this analysis, slide specimens were assigned to *M. sacchari* or *M. sorghi* according to their molecular assignment using SSR and EF1-α data.

## 177 Results

### 178 Morphometry

According to the unsupervised k-means clustering, the number of inferred morphological clusters was k = 2. The membership probability of each specimen is shown by Fig 1. The *M. sorghi* paratype was assigned to the morphological cluster 1, with a 100% membership probability.

Fig 1. DAPC analysis based on 11 morphological traits recorded on 51 slide-fixed specimens. Species identification by SSR and EF1-α: orange = *M. sacchari*, blue = *M. sorghi*.
187 Both clusters differed significantly (P < 0.0001) by their *HindTibia:pt* ratio. The blue
188 cluster (Fig 1), which contained the *M. sorghi* paratype and exhibited the lowest *HindTibia:pt*
189 (Fig 2 and S1 Table), was *M. sorghi*. The orange cluster (Fig 1), which exhibited the largest
190 *HindTibia:pt* ratio (Fig 2 & S2 Table), corresponded to *M. sacchari*.

191
192 **Fig 2. Comparison between M. sacchari and M. sorghi using the three traits showing the**
193 **highest loadings in the DAPC among 51 slide-fixed specimens.** The specimens are
194 assigned to *M. sorghi* or *M. sacchari* according to their SSR or EF1-α genotype.

195
196
197 The loading plot (Fig 3) showed that the morphological characters contributing most to
198 the DAPC were *pt:cauda*, *HindTibia:pt* and *pt:siph*. The graphical comparison of these three
199 main contributing traits between both species is presented in Fig 2. In these plots, the
200 assignment of the specimens to both species was made according to their SSR and EF1-α
201 genotype (see below). For the three traits, both species differ significantly with an uncorrected
202 P-value < 0.0001 (F = 100.3, 78.73 and 62.96 respectively for *pt:cauda*, *HindTibia:pt* and
203 *pt:siph*). However, some overlapping is observed for each trait.

204
205 **Fig 3. Canonical loading plot.** The horizontal line is the limit showing the variables
206 (morphological traits) that yield a cumulated 75% contribution to the DAPC. The individual
207 peaks show the magnitude of the influence of each variable on separation of *M. sorghi* and *M.
208 sacchari*.

209
210 The comparison of all morphological traits of *M. sorghi* and *M. sacchari* apterous
211 females is presented in S2 Table. There are significant differences between both species in
212 16 out of 34 traits.

213
214 The values for alatae are given in S3 Table. Due to the small number of specimens
observed, no statistical comparison was carried out and these values are only suggestive.
Because we only observed museum slide-fixed alate specimens, we could not genotype them. In the absence of a key for alatae, species assignment was carried out taking into account geographic origin, according to the worldwide repartition of both species (Fig 5). Specimens from West Africa (Burkina Faso, Ivory Coast and Senegal) were considered as *M. sorghi*, specimens from Reunion and Brazil were considered as *M. sacchari*. Although the number of alate specimens is insufficient to form a basis for any statistical comparison, it should be noted that, as in apterae, the *pt* of alatae assigned to *M. sacchari* tends to be longer than that of alatae assigned to *M. sorghi*, and that the ratios involving this parameter, especially *pt:siph*, may prove to be useful discriminants.

**Fig 5. Minimum spanning network constructed using the concatenated COI, COII and EF1-α sequences.** The orange and blue boxes indicate the STRUCTURE clusters inferred from SSR data (Table 4). The eight concatenated haplotypes (cH1 to cH8) are listed in Table 4. The number of hatch marks represents the number of mutations separating the concatenated haplotypes. Circle sizes are proportional to haplotype frequencies.

## 231 Microsatellites

Fifty-nine multilocus genotypes (MLG) have been identified (S6 Table). Fifty-six were published earlier [9,10,12] and three new ones (Ms25, Ms26, and Ms58) were observed during this study. The clustering with Structure, followed by the use of the Evanno et al. [31] method, leads to the conclusion that the number of inferred populations is *k* = 2 (S2 Fig).

The assignment of each MLG with *k* = 2 is presented in Fig 4. The first cluster groups MLL-A-E-F, the second cluster groups MLL-B-C-D.

**Fig 4. Assignment of each of the 59 MLG to the two clusters inferred by Structure.** MLG were defined using nine microsatellite markers.
The congruence between the DAPC morphological assignment to *M. sorghi* or *M. sacchari* and the Structure Bayesian clustering based on SSR genotyping showed unambiguously that the blue cluster is *M. sorghi* and the orange cluster is *M. sacchari* (Table 2). Among the 29 specimens belonging to the blue Structure cluster, 27 were morphologically assigned to *M. sorghi*, one was morphologically assigned to *M. sacchari* and one was undetermined. Among the 21 specimens belonging to the orange Structure cluster, 19 were morphologically assigned to *M. sacchari*, one was morphologically assigned to *M. sorghi* and one undetermined. The resulting accuracy of the congruence of Structure clustering and morphological clustering is 95.8% (46 / 48), when excluding the two undetermined specimens.

<table><caption>Table 2. Multi Locus Lineage (MLL) identification of the 51 slide fixed specimens morphologically assigned to *M. sorghi* or *M. sacchari* by DAPC.</caption><thead><tr><th rowspan="2">Structure cluster</th><th rowspan="2">MLL</th><th rowspan="2">level of MLL identification<sup>b</sup></th><th colspan="3">morphological assignment by DAPC</th></tr><tr><th><i>M. sacchari</i></th><th><i>M. sorghi</i></th><th>undetermined<sup>a</sup></th></tr></thead><tbody><tr><td rowspan="4">orange</td><td rowspan="2">MLL-C</td><td>specimen</td><td>1</td><td></td><td>1</td></tr><tr><td>sample</td><td>9</td><td><sup>1</sup><sub>c</sub></td><td></td></tr><tr><td rowspan="2">MLL-D</td><td>specimen</td><td>8</td><td></td><td></td></tr><tr><td>sample</td><td>1</td><td></td><td></td></tr><tr><td rowspan="4">blue</td><td rowspan="2">MLL-A</td><td>specimen</td><td></td><td></td><td></td></tr><tr><td>sample</td><td></td><td>16</td><td></td></tr><tr><td rowspan="2">MLL-F</td><td>specimen</td><td><sup>1</sup><sub>c</sub></td><td>7</td><td></td></tr><tr><td>sample</td><td></td><td>4</td><td>1</td></tr></tbody></table>

<sup>a</sup> The specimens with a DAPC membership lower than 0.8 are considered undetermined.

<sup>b</sup> MLL identification was carried out by SSR genotyping on the specimen itself or on other specimens from the same sample.

<sup>c</sup> Specimens whose molecular and morphometric assignments are discordant.
261 DNA sequencing

262 A total of 371 aphids were sequenced for at least one of three genes, COI, COII or
263 EF1-α: 340 for COI, 143 for COII, and 163 for EF1-α.

264 In COI (658 bp), we detected four SNPs defining four haplotypes (S4 Table). The
265 sequences were deposited in Genbank under accession numbers KJ083108-KJ083215,
266 KX453783-KX453784, MG838208-MG838315, MT813521-MT813656, and MT821458-
267 MT821474.

268 In COII (763 bp), we detected four SNPs defining five haplotypes (S5 table). The
269 sequences were deposited in Genbank under accession numbers MT847245-MT847387.

270 The EF1-α gene portion amplified was 1,014 bp long. The sequences were deposited
271 in Genbank under accession numbers MT847432-MT847594. Prior to analysis, the sequences
272 were trimmed to a 528 bp length (from position 248 bp to 775 bp), to discard low quality 5' and
273 3' sequence ends in most specimens. In this EF1-α 528 bp sequence portion, we detected 1
274 unambiguous SNP and 4 ambiguous positions (heterozygous) defining seven haplotypes (S6
275 Table). Only two haplotypes were defined when omitting the four ambiguous positions, and we
276 considered only these two haplotypes in further analysis. Haplotype H1 was only observed in
277 *M. sacchari* and haplotype H2 only in *M. sorghi*.

278 In CytB (745 bp), we detected one SNP defining two haplotypes. Both haplotypes were
279 detected in both *M. sacchari* and *M. sorghi*. The sequences were deposited in Genbank under
280 accession numbers MT847388-MT847423.

281 In the ITS1 region, we obtained 445-451 bp length sequences. These sequences
282 included a 3-6 bp indel region and one SNP. The two haplotypes defined by the SNP were
283 present in both *M. sacchari* and *M. sorghi* specimens. The sequences were deposited in
284 Genbank under accession numbers MT821305-MT821342.

285 In the ITS2 region, in a 462 bp-sequence length, we detected one SNP and three
286 indels, but the SNP was located within an indel and therefore could not be used in further
287 analysis. The sequences were deposited in Genbank under accession numbers MT821344-288 MT821448.

289 A total of 63 specimens were genotyped at the three genes COI, COII, EF1-α, and were 290 also successfully genotyped with SSR. The relationship between MLL and haplotypes among 291 these specimens is presented in Table 3. EF1-α provided a diagnostic substitution at position 292 637 separating *M. sorghi* from *M. sacchari*. The distinctive base was T for *M. sacchari* and A 293 for *M. sorghi*. COI provides an incomplete separation of both species: haplotypes H2 and H3 294 are diagnostic of *M. sacchari*, H6 is diagnostic of *M. sorghi*, but haplotype H1 is present in both 295 species.

<table><caption>296 Table 3. Haplotypes defined by the concatenation of four genes and correspondence 297 with the Multilocus Lineages (MLLs) defined using SSR.</caption><thead><tr><th rowspan="2">concatenated haplotype</th><th colspan="3">gene / haplotype</th><th rowspan="2">species</th><th rowspan="2">MLL</th><th rowspan="2">Structure Cluster</th><th rowspan="2">n</th></tr><tr><th>CO1</th><th>CO2</th><th>EF</th></tr></thead><tbody><tr><td>cH1</td><td>H1</td><td>H3</td><td>H2</td><td>sorghi</td><td>A</td><td>1</td><td>18</td></tr><tr><td>cH1</td><td>H1</td><td>H3</td><td>H2</td><td>sorghi</td><td>F</td><td>1</td><td>8</td></tr><tr><td>cH2</td><td>H1</td><td>H3</td><td>H1</td><td>sacchari</td><td>B</td><td>2</td><td>2</td></tr><tr><td>cH3</td><td>H2</td><td>H1</td><td>H1</td><td>sacchari</td><td>C</td><td>2</td><td>11</td></tr><tr><td>cH4</td><td>H2</td><td>H5</td><td>H1</td><td>sacchari</td><td>C</td><td>2</td><td>5</td></tr><tr><td>cH5</td><td>H3</td><td>H3</td><td>H1</td><td>sacchari</td><td>D</td><td>2</td><td>10</td></tr><tr><td>cH6</td><td>H1</td><td>H2</td><td>H2</td><td>sorghi</td><td>E</td><td>1</td><td>6</td></tr><tr><td>cH7</td><td>H6</td><td>H3</td><td>H2</td><td>sorghi</td><td>F</td><td>1</td><td>1</td></tr><tr><td>cH8</td><td>H1</td><td>H4</td><td>H2</td><td>sorghi</td><td>A</td><td>1</td><td>2</td></tr></tbody></table>

298 The number of variable sites was nine on the 1,806 bp of the three concatenated gene 299 sequences (i.e. 0.49%), defining eight haplotypes, with a nucleotide diversity of 0.13%. The 300 minimum spanning network among haplotypes is shown by Fig 5. *M. sorghi* specimens defined 301 a star shaped haplogroup centered on haplotype cH1. *M. sacchari* defined a less 302 homogeneous haplogroup, each haplotype being separated from the other by two or three 303 substitutions. 304
306

The within species and between species divergences for the six sequenced gene portions are summarized in Table 4.

307

308

<caption>**Table 4. Sequence divergences (pairwise uncorrected P-distances, %) between or within species.**</caption>

309

<table><thead><tr><th></th><th>COI</th><th>COII</th><th>EF1α</th><th>CytB</th><th>ITS2</th><th>ITS1</th></tr></thead><tbody><tr><td>within <i>M. sorghi</i></td><td>0.15<br>(0.15 - 0.15)<br>n = 184</td><td>0.18<br>(0.13-0.26)<br>n = 70</td><td>0<br>-<br>n = 88</td><td>0<br>-<br>n = 11</td><td>0<br>-<br>n = 67</td><td>0.16<br>(0 - 0.26)<br>n = 18</td></tr><tr><td>within <i>M. sacchari</i></td><td>0.30<br>(0.30 - 0.30)<br>n = 136</td><td>0.18<br>(0.13-0.26)<br>n = 73</td><td>0<br>-<br>n = 83</td><td>0.13<br>(0.13 - 0.13)<br>n = 32</td><td>0<br>-<br>n = 38</td><td>0.13<br>(0 - 0.26)<br>n = 20</td></tr><tr><td>between species</td><td>0.28<br>(0 - 0.45)<br>n = 320</td><td>0.18<br>(0 - 0.26)<br>n = 143</td><td>0.19<br>(0.19 - 0.19)<br>n = 171</td><td>0.09<br>(0 - 0.13)<br>n = 43</td><td>0<br>-<br>n = 105</td><td>0.13<br>(0 - 0.26)<br>n = 38</td></tr></tbody></table>

310

Values are the mean of the pairwise distances; minimum and maximum distances for these comparisons are given in parentheses. Number of genotyped specimens = n. Species identification was carried out by SSR genotyping on the specimens themselves or on other specimens from the same sample.

311

312

313

314

315

316

## Geographical distribution of *M. sacchari* and *M. sorghi*

317

The geographical distribution of *M. sacchari* and *M. sorghi*, is presented in Fig 6 and is based on 2,332 genotyped specimens. Specimens belonging to MLL-A-E-F were assigned to *M. sorghi*, while specimens belonging to MLL-B-C-D were assigned to *M. sacchari*. In the absence of SSR genotyping, specimens bearing the EF1-α haplotype H1 or the COI haplotypes H2 or H3 were assigned to *M. sacchari* while specimens bearing EF1-α haplotype H2 were assigned to *M. sorghi* (Table 4). The remaining specimens (n = 24) were considered unidentified and therefore not taken into account in Fig 6. Only one unambiguous (i.e. allowing the species identification) data point from India was available from a public database (see Fig 6 legend). Most COI sequences stored in these public databases are either haplotypes that

318

319

320

321

322

323

324

325
326 we did not encounter in our study or are H1 haplotype, which is uninformative because it is
327 shared by *M. sorghi* and *M. sacchari*.

328

329 **Fig 6. Molecular identification with SSRs and sequencing of COI or EF1-α of 2,332**
330 **specimens:** blue = *M. sorghi*, orange = *M. sacchari*. Data from India is the EF1-α sequence
331 Genbank accession KU048048.1 (exact geographical location within India not available).

332

333

334 In West and in Southern Africa, *M. sorghi* is the sole detected species. In East Africa,
335 *M. sacchari* has been detected in Kenya and Tanzania, and *M. sorghi* in Uganda and Kenya.
336 In Kenya, both species coexist and were collected in the same sample once. Reunion and
337 Mauritius, in the South West Indian Ocean, are exclusively colonized by *M. sacchari*. The
338 Neotropical zone is colonized by *M. sacchari*. The Nearctic zone is colonized by both species,
339 as a result of the recent introduction of *M. sorghi* in the Americas. In Asia, *M. sorghi* is present
340 in China and India, and *M. sacchari* in Cambodia. In Australia and in Hawaii, only *M. sacchari*
341 was detected.

341

342

Discussion

343 Genetic analyses with SSRs and three gene sequences showed that two genetic
344 clades exist, one grouping MLL-A-E-F and the other grouping MLL-C-D. The multivariate
345 morphometric data analysis separated the specimens in two groups matching the two genetic
346 clades. Comparison with a *M. sorghi* paratype and comparison of the *HindTibia:pt* ratio [6,7,26]
347 confirmed that the genetic clade grouping MLL-A-F is *M. sorghi* while the clade grouping MLL-
348 C-D is *M. sacchari*. The status of MLL-B (Australian specimens) remains to be confirmed: it is
349 assigned to *M. sorghi* by SSRs and EF1-α sequence, but we did not confirm this assignment
350 by morphometric means, due to a lack of specimens. Similarly, the lack of specimens
351 prevented us from studying the morphology of MLL-E (all from China) and its taxonomic status

343

344

345

346

347

348

349

350

351
352 remains uncertain, although SSRs and EF1-α sequence data both suggest it belongs to M. 353 sorghi.

354 Three morphological criteria are useful for species separation: pt:cauda, pt:siph and 355 HindTibia:pt. However, as observed by Blackman and Eastop [7,26], there are no clear limits 356 between species and values overlap largely (Fig 2). The ranges for M. sacchari vs. M. sorghi 357 are respectively pt:cauda (1.72-2.35) vs. (2.19-2.8), HindTibia:pt (1.8-2.31) vs. (1.54-1.93) and 358 pt:siph (2.71-4.28) vs. (2.26-3.19). Due to overlap, the use of these morphological criteria can 359 lead to ambiguous results and should be applied quantitatively (i.e. at the population level) 360 rather than qualitatively (i.e. at the individual level).

361 The molecular diagnostic methods for separation of M. sacchari and M. sorghi are 362 summarized in Figs 7 and 8. COI can allow for the identification of M. sacchari through two 363 specific SNPs in positions 263 or 294. But, because haplotype H1 (Table 3) is shared by both 364 species, an unambiguous identification of M. sorghi with COI is sometimes not possible. With 365 EF1-α, a specific SNP in position 637 allows the separation of both species. Genotyping with 366 a single SSR locus also allows the separation of both species. For example, the SSR locus 367 CIR-Ms-G01 can be used for this purpose (Fig 8). The two alleles present at this locus in M. 368 sacchari are separated by 25 to 31 bp, whereas M. sorghi genotypes are mostly homozygous 369 or exhibit two alleles separated by 4 to 8 bp only (S7 table).

370

371 **Fig 7. Molecular diagnosis for separation of M. sacchari and M. sorghi using sequencing** 372 of COI or EF1-α.

373 **Fig 8. Molecular diagnosis for separation of M. sacchari and M. sorghi using the SSR** 374 locus CIR-Ms-G01. M. sacchari (MLL-D) are in lanes A10 ( voucher # SNIB00040_0101) and 375 A1 ( voucher # SNIB00233_0102). M. sorghi (MLL-F) are in lanes A11 ( voucher # 376 SNIB00075_0101) and A5 ( voucher # SNIB000237_0102). The PCR was carried out 377 according to [14]. The migration of PCR products was carried out on a Qiagen Qiaxcel 378 electrophoresis analyzer. The image was generated by the Qiaxcel ScreenGel 1.6.0 software.

379
380

We observed low genetic distances between *M. sacchari* and *M. sorghi* when comparing gene sequences that are widely used in aphid taxonomy. A 'borderline' distance between species in the COI barcode region is sometimes used by some authors to confirm species limits [32,33]. However, it is now well recognized that there is no universal genetic distance separating aphid species and that low genetic difference between species can occur in aphids. Multiple examples of low COI, COII, CytB pairwise distance between species exist [34-36]. For example, a situation very similar to ours occurs in the genus *Megoura*, where Kim and Lee [37] observed an absence of COI, COII, CytB, ITS1, ITS2 divergence between *Megoura litoralis* Müller and *Megoura viciae* Buckton, which differ by a 0.2% P-distance on EF1-α.

According to Blackman et al. [6], *M. sorghi* is more likely observed on sorghum and *M. sacchari* more likely on sugarcane, although not absolutely specific to the hosts indicated by their names. In our study, the samples were distributed as follows: 14 from sugarcane vs. 94 from sorghum (all *Sorghum* species together) for *M. sorghi*, and 18 samples from sorghum vs. 69 from sugarcane for *M. sacchari*. Although our sampling plan was not designed to test host plant preference, the difference of preference between the two species appears obvious. The results of Boukari et al. [38] obtained in Florida confirm this preference, showing that sugarcane harbors almost only *M. sacchari* (COI haplotypes H2 and H3), which is absent from aphids on *Sorghum* spp. The recent work by Paudyal et al. [39] in the USA also supports this apparent preference. Indeed, using host transfer experiments, these authors have demonstrated that MLL-F strains collected from *Sorghum* spp. exhibited a higher fitness on sorghum than on sugarcane, and that an MLL-D strain collected from sugarcane exhibited a higher fitness on sugarcane than on sorghum. According to this host preference difference, we suggest that the common name 'sugarcane aphid' should be used for *M. sacchari* and 'sorghum aphid' for *M. sorghi*.

Our study shows that the invasive genotype responsible for outbreaks on sorghum in North and Central America and the Caribbean islands since 2013 is MLL-F [12], which, belongs
408 to *M. sorghi*, while the genotype present before 2013 (MLL-D) is *M. sacchari*. If the hypothesis
409 of a lower fitness of *M. sacchari* on sorghum compared to *M. sorghi* is confirmed, this would
410 explain why no damage was observed on sorghum prior to the introduction of *M. sorghi* to the
411 Americas.

412 Acknowledgements

413 We are grateful to Hughes Telismart, Magali Hoarau and Antoine Franck for technical
414 assistance. We also thank Susan E. Halbert for her helpful comments on this manuscript. We
415 also acknowledge Armelle Coeur d'Acier (CBGP), Laurent Fauvre and Thierry Bourgoin
416 (MNHN) for providing access to the CBGP and MNHN collections.

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516

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517

Aphididae) genotypes. Trends in Entomology 15: 47-58.

518

Supporting information

519

S1 Fig. *Melanaphis* apterous female habitus.

520

S2 Fig. Evanno method inferring k = 2.

521

S1 Table. List of samples and specimens.

522

S2 Table. Comparison of morphological characteristics of *M. sacchari* and *M. sorghi* apterous females.

523

524

S3 Table. Comparison of morphological characteristics of *M. sacchari* and *M. sorghi* viviparous alate females.

525

526

S4 Table. CO1 haplotypes, position and nature of nucleotide substitutions.

527

S5 Table. CO2 haplotypes, position and nature of nucleotide substitutions.

528

S6 Table. EF1-α haplotypes, position and nature of nucleotide substitutions.

529

S7 Table. Observed microsatellite Multi Locus Genotypes (MLG).

530
<figure><img src="image_3.png" /><figcaption>Fig 1</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Fig 2</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Fig 6</figcaption></figure>
ATAATATTAGATTTTGA**Y**TTTTACCTCCATCATAATAATAATTT**R**TAGATTTATAATTA

<figure><img src="image_6.png" /></figure>

T = *M. sacchari*

C = non-specific

<figure><img src="image_7.png" /></figure>

A = *M. sacchari*

G = non-specific

EF1-alpha

TTTTTATGAT**W**TGTAGTTTCTAATTTTAATTTATTT**R**TAGG

<figure><img src="image_8.png" /></figure>

T = *M. sacchari*

A = *M. sorghi*

A/G/R (homozygous or heterozygous)
non-specific
bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.351833; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

Loadings

<figure><img src="image_9.png" /><figcaption>Fig 3</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Fig 4</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Fig 5</figcaption></figure>
<figure><img src="image_12.png" /></figure>

bioRxiv preprint doi: https://doi.org/10.1101/2020.10.23.351833; this version posted October 23, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.

<caption>Fig 8</caption>