Patterns in the alien flora of the Democratic Republic of the Congo: a comparison of Asteraceae and Fabaceae

Background and aims – This work provides the first pattern analysis of the alien flora of the Democratic Republic of the Congo (D.R. Congo), using Asteraceae and Fabaceae as a case study. Methods – Based on herbarium collections, existing databases, and literature data, a database of 38 alien species of Asteraceae and 79 alien species of Fabaceae has been assembled. Patterns in the introduction pathway, phylogeny, life form, morpho-functional traits, geographic origin, and occurrence in D.R. Congo are explored. Key results – America is the main source continent in both families, but Asia is also an important donor of Fabaceae. Taxonomic spectrum discrepancies between the alien and the native flora reflect the continent of origin. Sixty-six percent of alien Asteraceae have been accidentally introduced, most of which being annual weeds of disturbed soil. In contrast, 90% of alien Fabaceae have been deliberately introduced for forestry, agriculture, or environmental purposes, most of which being phanerophytes. Traits were compared between pairs of congeneric alien and native species. For Asteraceae, a sharp discrepancy was found in the life form spectrum (aliens: mostly therophytes; natives: phanerophytes). For Fabaceae, alien species had larger leaves and larger pods compared to their native congeners. The number of specimens in collections was positively correlated with the time since the date of first collection for both families. The GuineoCongolian region has the highest number of alien Fabaceae, while alien Asteraceae are overrepresented in the Zambezian region. Conclusions – Contrasting patterns between alien Asteraceae and Fabaceae in the flora of D.R. Congo in terms of life forms, trait divergence compared to the native flora, and occurrence, reflect the divergent biological attributes and relations to humans of the two families. The striking discrepancies between the two families call for analyses of patterns of alien flora at family level and warn against global generalisations.


INTRODUCTION
Non-native organisms represent an ever-increasing proportion of the biota worldwide (van Kleunen et al. 2015;Pyšek et al. 2017;Turbelin et al. 2017;Seebens et al. 2018;Essl et al. 2019) and the problem of alien species has become a global conservation issue (Mack et al. 2000;Lambertini et al. 2011;McGeoch et al. 2016).
Compiling alien flora inventories is an essential step to initiate monitoring and assess the impact of alien species (Pyšek et al. 2004;Randall et al. 2008;McGeoch et al. 2016;Latombe et al. 2017). Exploring patterns in the alien flora of a territory can provide insights into the origin and pathways of introduction of aliens . The alien and the native flora often have a distinct phylogenetic assemblage, which has implications for interactions with the resident biota (Divíšek et al. 2018). Pattern analysis in such inventories also helps prioritise management actions (Randall et al. 2008;McGeoch et al. 2016). Pattern analysis should include species traits and introduction characteristics (Dawson et al. 2011). Comparison of functional traits between the alien and the native flora has provided key insight into the biological attributes underlying establishment, naturalisation, and invasion success (Pyšek & Richardson 2007;Ordonez et al. 2010;van Kleunen et al. 2010van Kleunen et al. , 2014Gallagher et al. 2015;Moravcová et al. 2015;Divíšek et al. 2018). However, the factors explaining the success of alien plant species, including propagule pressure and residence time, are different at different stages of the invasion (Dawson et al. 2009(Dawson et al. , 2011. Many tropical African countries suffer from a lack of alien species inventories (but see Maroyi 2012;Rejmánek et al. 2016;Noba et al. 2017;Witt et al. 2018;Ansong et al. 2019). They seem to host relatively low numbers of alien species Turbelin et al. 2017) possibly due to lower levels of anthropogenic pressure but also due to data deficiency (Pyšek et al. 2008;Essl et al. 2019).
The Democratic Republic of the Congo (further D.R. Congo) represents a major hotspot of biodiversity in Africa (Sosef et al. 2017). The native flora of D.R. Congo is estimated to comprise ca. 11,000 species with 18.3% endemism Sosef et al. 2017). The forests of the Congo Basin (rainforest and dry tropical woodland) provide services of key importance to the sustainable development of Central Africa and the planet, including biodiversity conservation, carbon storage, climate stabilization, soil protection, and provisioning of natural resources (Kamdem-Toham et al. 2003;Harrison et al. 2016). However, the Congo Basin is subjected to steadily increasing human influence due to deforestation and urbanisation (Anonymous 2012;Potapov et al. 2013). Increasing human pressure could favour the expansion of non-native species (Mack et al. 2000;Kowarik 2003;Essl et al. 2019). In spite of its crucial importance for D.R. Congo to plant diversity conservation in tropical Africa, the information on alien plant species is extremely scarce (Boy & Witt 2013;Latombe et al. 2017), with very few case studies in the last decade (Zachariades et al. 2013;Useni Sikuzani et al. 2018;Mbale et al. 2019). Pyšek et al. (2017) reported 522 naturalized species in D.R. Congo, i.e., 4.5% of the total flora.
In this paper, we explore patterns in the alien flora of D.R. Congo for the first time. We focus on Asteraceae and Fabaceae, i.e., the two largest families in terms of native and naturalized species in sub-Saharan Africa (Klopper et al. 2007) and worldwide . Along with Poaceae, they are the three families most represented among successful invaders (Randall 2017). The comparison of those two families is particularly interesting because they have contrasting biological traits (nitrogen fixator vs. non-fixator), dispersal strategies (Asteraceae are often wind-dispersed), and relations to humans, with Fabaceae being widely used in agroforestry and agriculture Binggeli 2011).
We first assemble a checklist of alien species for the two families. Secondly, we examine the life form spectrum, geographic origin, introduction pathway, and taxonomic assemblage of the alien species. Thirdly, we compare alien and native species for life forms and traits, correcting for phylogenetic bias by using pairs of congeners. Lastly, we examine occurrence (expressed as the number of specimens) and alien species richness distribution among the different phytogeographic districts of D.R. Congo. We anticipate contrasting patterns between the two families, due to contrasting biological attributes and relations to humans.

Study area
D.R. Congo covers 2,345,409 km 2 in Central Africa, spanning from 13°S to 5°N, and harbours at least five types of climate (according to the Köppen classification; Peel et al. 2007), i.e., tropical rain forest (Af), tropical monsoon (Am), tropical wet and dry (Aw), temperate with dry winter and hot summer (Cwa), temperate with dry winter and warm summer (Cwb). The vegetation of D.R. Congo is highly diversified depending on climate and phytogeographic context. White's (1983) phytogeographic system recognizes six phytochoria in D.R. Congo, i.e., four regional centres of endemism (RCE) (the Guineo-Congolian RCE, the Zambezian RCE, the Sudanian RCE, and the Afromontane archipelago-like RCE) and two transition zones, i.e., the Guineo Congolian/Zambezian regional transition zone and the Guinea-Congolia/Sudania regional transition zone. Robyns (1948) divided D.R. Congo into 10 phytogeographic districts, based on vegetation cover and flora. This phytogeographic system is no longer fully satisfying, but it is still in use in floristic publications because herbarium collections are managed accordingly.

Data assemblage
Species considered in this work are alien species that have been observed out of cultivation in D.R. Congo. This definition includes casual, naturalized (= established), and invasive species (Blackburn et al. 2011).
D.R. Congo entries in the Global Register of Introduced and Invasive Species (GRIIS, http://www.griis.org)   The status (native/alien) of a significant number of species is not easy to ascertain, in particular for species with a pantropical distribution, for which the region of origin is often difficult to trace (Binggeli et al. 1998;Gallagher 2016;Essl et al. 2019 Only records based on herbarium vouchers have been considered. Therefore, due to the lack of herbarium material, the following species in the GRIIS and GLoNAF lists have been excluded from the database, i.e., Asteraceae: Artemisia annua, Flaveria trinervia, Gnaphalium pensylvanicum, Montanoa hibiscifolia, Tagetes erecta, Youngia japonica; Fabaceae: Acacia decurrens, Aeschynomene americana, Cassia eremophila, Cassia surattensis subsp. glauca, Gliricidia sepium, Leucaena diversifolia, Mimosa invisa, Sesbania punicea, Vigna juruana. One species has been excluded due to unresolved synonymy (Tagetes lucida).
Ultimately, the database comprises 38 and 79 alien species for Asteraceae and Fabaceae, respectively (supplementary files 1 and 2). In total, 663 and 1138 native species were retrieved from the digital Flora of Central Africa for Asteraceae and Fabaceae, respectively.

Taxonomic spectrum, traits, and life form
Each species was assigned to a subfamily and tribe based on LPWG (2017) for Fabaceae and Fu et al. (2016) for Asteraceae. The taxonomic assemblages (i.e., tribe frequency distribution) of native and alien species were compared using Chi-squared tests.
For trait values, our primary source of information is the digital Flora of Central Africa. For some missing data, other databases were used, i.e., Plants of the World Online, Plant Resources of Tropical Africa (PROTA) (www.prota.org), and World Agroforestry (www.worldagroforestry.org).
For life form, Raunkiaer's classification was followed but, in a few cases, we had to make somewhat arbitrary decisions including, i) large lianescent plants with herbaceous pluriannual shoots renewed from the rootstock, and ii) robust shrub-like forbs with shoots renewed from rhizome at the onset on the rain season but persisting for several years if not destroyed by fire and re-sprouting from lateral buds. Both were treated as geophytes.
The following morpho-functional traits were recorded for all alien species, based mostly on the digital Flora of Central Africa. These traits capture different functional dimensions. For both families: height (m), leaf length and width (cm) (correlated to light capture strategy and competitive ability); for Fabaceae with compound leaves, individual leaflets were considered; for Asteraceae: flower head (capitulum) diameter (mm) (a proxy of floral display), achene length (mm), pappus length (mm) (related to reproductive strategy, propagule pressure, and dispersal ability); for Fabaceae: vexillum size (mm) or, alternatively, flower head diameter (mm) for species with clustered flowers (e.g., Mimosa spp.) (proxy of floral display), rachis length (cm) in compound leaves and petiole length (cm) in simple leaves, pod length (cm), seed length (mm) (related to reproductive strategy, propagule pressure, and dispersal ability). The upper and lower values of the variation range have been recorded from the abovementioned floras, and the mean value was used in statistical tests.

Introduction pathway
Any alien species that is currently planted or cultivated (crops, ornamentals, forestry, and landscape) anywhere in D.R. Congo or elsewhere in tropical Africa has been considered as intentionally introduced. Our primary sources of information are the digital Flora of Central Africa, Plants of the World Online, Plant Resources of Tropical Africa (PROTA), and World Agroforestry.
Intentional introductions have been categorized as follows: ornamental, agriculture (including agroforestry, fodder, environmental purposes (soil fertilizer, etc.)), edible, for- The list includes only those species that have been found out of cultivation and for which herbarium vouchers are available. Date = date of the most ancient specimen; n = total number of specimens.

Comparison of congeneric alien and native species
Trait comparison between the native and the alien flora is subjected to phylogenetic bias because the two groups have different phylogenetic/taxonomic compositions. To overcome this bias, trait comparisons were performed between congeneric alien and native species. Congeneric species pairs have been successfully used in the comparative biology of alien species (Grotkopp & Rejmánek 2007;Pyšek & Richardson 2007;van Kleunen et al. 2010;Godoy et al. 2011;Gallagher et al. 2015).

Selection of congeneric pairs
The database was screened for genera comprising both native and alien species in D.R. Congo. Some genera comprised more than one alien and/or native species. In such cases, the selection of congeneric species pairs was based on the most recent infrageneric phylogeny or taxonomy. As a rule, the native species most closely related to the alien species were selected. When several species met the selection crite-rion, a "pseudospecies" was created by calculating the average of trait values of all the species in the group (Asteraceae: Bidens (natives), Erigeron (aliens), Gynura (natives), Mikania (natives), and Sonchus (aliens and natives); Fabaceae: Canavalia (aliens), Cassia (aliens), Clitoria (natives), Erythrina (natives), Vigna (natives), and Zornia (natives)). For the comparison of life form, a nominal trait, "pseudospecies" could not be computed and all congeneric species fulfilling the selection criteria were included. For Asteraceae, as only seven genera comprised both native and alien species, one intergeneric comparison within the same tribe was also included (i.e., tribe Eupatorieae: Ageratum conyzoides (alien) and all native Mikania species). Eight and 18 phylogenetically independent comparisons fulfilled the selection criteria for Asteraceae and Fabaceae, respectively (supplementary files 3 and 4). The same traits were recorded for congeneric natives as for the aliens (see above), using the same sources of information.
Continuous traits were compared using Student's t-tests on paired data or its non-parametric counterpart Wilcoxon signed rank test. The life form spectrum of native and alien species was compared using Fisher's exact test.

Number of specimens and occurrence
The distribution within D.R. Congo was recorded as presence/absence in Robyns' (1948) phytogeographic districts based on exhaustive screening of the collections at BR.
The number of herbarium specimens in the collections at BR has been used as a proxy for the extent of occurrence of the species in D.R. Congo, assuming that more widespread species tend to be more often collected, everything else being equal (Stadler et al. 1998;Crawford & Hoagland 2009;Maroyi 2012). Alien species can differ in their extent of occurrence due to different times since introduction (Seebens et al. 2018). The date of the introduction of alien species in D.R. Congo is not known. Instead, the collection date of the most ancient specimen in the collections has been used as a proxy. Establishment success and range size can also be influenced by traits. Multiple regressions were used to examine if the number of specimens (dependent variable) is correlated to time since the first collection and to morpho-functional traits (independent variables).
A total of 74% of alien Asteraceae originated from America, far ahead of all other continents (3-8% each) ( fig. 2). Sixty-six percent of aliens have been introduced to D.R. Congo accidentally. Most deliberate introductions correspond to ornamental species (24% of aliens) ( fig. 3). Therophytes accounted for 71% of alien Asteraceae, far more than all other life forms ( fig. 4). Number of specimens and distribution within D.R. Congo -The alien species with the oldest herbarium specimens from D.R. Congo are Ageratum conyzoides (first collection in 1812), Taraxacum officinale (1847), and Bidens pi-          . 7). Phanerophytes are much less represented in aliens (8%) compared to natives (41%), while therophytes are the most frequent life form in alien Asteraceae (75% vs. 18% in natives). No significant difference was found for any quantitative trait between native and alien Asteraceae (table 2).
Therophytes are slightly less frequent in alien Fabaceae (14%) compared to congeneric natives (17%). However, the life form spectra of the two groups are not significantly different (Fisher's exact test: P = 0.71) ( fig. 9). Congeneric natives and aliens were significantly different for three quantitative traits, i.e., rachis length, leaflet length, leaflet width, and pod length. Alien species had ca. 61% longer rachis, 64% longer and 66% wider leaflets compared to their native congeners, and ca. 59% longer pods (table 2).

DISCUSSION
To the best of our knowledge, our work represents the first assessment of the alien flora of D.R. Congo in terms of taxonomic assemblage, morpho-functional traits, pathway of introduction, region of origin, and occurrence. The approach is focused on Asteraceae and Fabaceae, the two largest families in the flora of D.R. Congo, representing together 22.4% of the total alien flora.
D.R. Congo does not appear to host large numbers of alien plant species in comparison to other tropical African countries (table 3). This is surprising considering that D.R. Congo is the largest tropical African country. Tropical Africa, in general, does not appear to be a hotspot of alien species (van Kleunen et al. 2015;Turbelin et al. 2017). Following Foxcroft et al. (2010), the lower extent of alien plant invasions in African savannas is attributable to lower rates of intentional plant introductions, the role of large mammalian herbivores, and the adaptation of African systems to fire. Tropical savannas may also be relatively resistant to invasion due to resource limitation (Taylor et al. 2018). The relatively low economic development is another factor explaining low levels of biological invasions ).
It is also quite possible that our database is incomplete. Although the flora of D.R. Congo is well represented in collections (5629 specimens for the 117 alien Asteraceae and Fabaceae species), alien species records are scarce for the last decades. Of the 117 alien species in this study, as few as 35 (13 Asteraceae and 22 Fabaceae) have been collected after 2000 (supplementary files 1 and 2). Therefore, the collections do certainly not provide an up to date picture of the alien flora in D.R. Congo, recent introductions certainly being underrepresented. This limitation must be kept in mind when interpreting our results.

Contrasting patterns between Fabaceae and Asteraceae
A striking result is the discrepancy between alien Asteraceae and Fabaceae for most of the investigated features. The differences in the introduction pathway, life forms, traits, and distribution are mutually consistent, in line with divergent biological attributes and relations to humans between the two families.
Geographic origin of aliens -Asteraceae and Fabaceae in D.R. Congo primarily originate from the Americas (74% and 54%, respectively). This result confirms previous work pointing to the prominent contribution of the Americas to the alien flora of Africa, due to a legacy of a long history of trade and exchange between tropical America and Africa, starting long before colonisation (Chevalier 1931;Wild 1968;Roussel & Juhe-Beaulaton 1992;Katz 1998;Maroyi 2012;Witt et al. 2018;Ansong et al. 2019). Such exchanges may explain both accidental introductions (mostly Asteraceae, e.g., Ageratum conyzoides and Bidens pilosa) and deliberate introductions of crops and other edible species (mostly Fabaceae; e.g., Canavalia ensiformis and Phaseolus lunatus). Interestingly, the contribution of Asia as a region of origin is much larger for Fabaceae compared to Asteraceae, probably due to relatively recent deliberate introductions for agriculture and   agroforestry (e.g., Pterocarpus indicus (first record: 1936) and Crotalaria spectabilis (1933)). The prominent contribution of America to alien Asteraceae and Fabaceae is reflected in the distinct phylogenetic assemblage of the alien flora, with Heliantheae (8 species) and Cassieae (16 species), two mostly American tribes, overrepresented among aliens. Introduction pathway -Most of the alien Fabaceae (71 of 79, i.e., 90%) have been intentionally introduced to D.R. Congo primarily for forage, fertilizer, and agroforestry (38%). The high proportion of phanerophytes amongst alien Fabaceae (59 of 79, i.e., 75%) can be explained by the deliberate introduction for agri-environmental purposes as the main source of successful aliens in the flora of D.R. Congo. Intentional introduction facilitates the spread of alien species . Silviculture and agroforestry are major pathways of alien species introduction, and both have contributed noxious invasive woody species in the tropics (Richardson 1998;Richardson et al. 2004;Binggeli 2011). Our results show that the Forestier Central district, mostly in Guineo-Congolian rainforest, hosts a particularly large number of alien Fabaceae. This possibly points to a role of botanical gardens and forestry experimental stations in the introduction of those legumes, as two important agronomic and botanical research centres of the Belgian colony (Yangambi and Eala, both with botanical gardens and nurseries) were situated in the Forestier Central district (Binggeli et al. 1998). Tropical botanical gardens are known to be an important source of alien plant naturalisation in Africa (Dawson et al. 2008).
The pattern is quite different for Asteraceae, with 66% of alien species introduced accidentally. This is consistent with the large proportion of therophytes among alien Asteraceae. Alien Asteraceae are mostly annual weeds on disturbed ground. The two most collected species are Ageratum conyzoides and Bidens pilosa, which both have spread quickly in D.R. Congo due to their efficient dispersal mechanisms (anemochory and epizoochory, respectively) while remaining restricted to waste ground and fallow fields. The large number of alien Asteraceae in Haut-Katanga can be interpreted in this context because short cycling Asteraceae are well-adapted to the strongly seasonal climate of the Zambezian region. Masocha et al. (2011) showed that alien Asteraceae are favoured by frequent burning in Zambezian savannas. Most of the deliberately introduced Asteraceae species were imported for ornamental purposes, and some of them have successfully naturalised (e.g., Tithonia diversifolia in SE D.R. Congo). In other parts of tropical Africa, ornamentals in amenity gardens are also a major source of alien plant introductions (Foxcroft et al. 2008;Bigirimana et al. 2012). However, such garden escapes often appear to be restricted to road verges and other human transformed habitats probably because of their high requirements for nutrients (Witt et al. 2019). Traits -We compared congeneric alien and native species in order to avoid bias due to different taxonomic assemblages in alien and native flora. For Asteraceae, aliens comprised a much larger proportion of therophytes compared to native species. This suggests that alien Asteraceae do not occupy the same habitats as their native congeners. As previously discussed, most alien Asteraceae in D.R. Congo are weeds on disturbed ground. Such species probably do not represent a major threat to native biota of high conservation value in D.R. Congo. In contrast, there were no differences for continuous traits between alien and native Asteraceae. Dawson et al. (2011) insists that traits do not play a prominent role in explaining the success of alien tropical plants compared to propagule pressure, for instance. It is also possible that traits not considered in the present work account for the success of alien species, including specific leaf area (SLA) and other traits related to resource capture and use (Grotkopp & Rejmánek 2007;Pyšek & Richardson 2007;van Kleunen et al. 2010;Moravcová et al. 2015).
The pattern was quite different for Fabaceae with phanerophytes being the most frequent life form in both aliens and natives. Fabaceae are one of the largest families in a broad range of forest and woodland types in D.R. Congo (Lebrun & Gilbert 1954). Most of the alien Fabaceae are trees and shrubs that have been introduced for agroforestry and landscape purposes, a well-known introduction pathway for noxious aliens elsewhere in Africa (Richardson 1998;Richardson et al. 2004;Binggeli 2011). The Forestier Central district, which concentrates the largest number of alien legumes, corresponds to the rainforest region of D.R. Congo. Thus, in contrast to Asteraceae, congeneric alien and native legumes could co-occur in the same vegetation types. Implications for conservation management need to be investigated.
Interestingly, alien legumes tend to be bigger than congeneric natives, significantly so for rachis, leaflet size, and pod length. This could be explained by a strong filter acting during the establishment, most likely due to the intentional selection of alien species with a syndrome of increased vigour for agroforestry (most species) and subsistence agriculture (e.g., Canavalia ensiformis and Vigna radiata). It would be interesting to test if larger leaf size implies higher competitive ability in alien species. While having larger fruits, alien Fabaceae do not have larger seeds, suggesting that larger pods could translate into larger seed output, itself a strong determinant of the rate of spread of alien species (Dawson et al. 2011;Moravcová et al. 2015).
Introduction and large-scale cultivation of alien woody species without weed risk assessment is in conflict with sustainable development (Richardson 1998). In this regard, it is particularly worrying that an assessment of forest genetic resources in D.R. Congo by the FAO (Malele Mbala 2003) failed to discriminate between native species of high conservation value and alien species, recommending conservation of Acacia auriculiformis, Albizia lebbeck, Cassia siamea, and Leucena leucocephala, four notoriously noxious alien species. Establishment and success of alien species -There is extensive variation among alien species in the number of specimens held in herbarium collections (from 1 to 553). Much of that variation is explained by the time elapsed since the most ancient collecting date. Records increased at a similar rate with time in the two families, suggesting that time since introduction is the driver of that relationship. However, while an early specimen obviously implies early introduction, species with more recent first records are not necessarily more recent introductions. Variation in record numbers can also be due to species-specific time-lags (Binggeli et al. 1998) and could indicate that different species have reached different stages in the invasion process (Blackburn et al. 2011). Species with the largest numbers of records can be safely considered as naturalized, and some of them possibly as invasive (e.g., Asteraceae: Ageratum conyzoides, Bidens pilosa, Chrysanthellum indicum, Erigeron bonariensis, Eclipta prostrata, Synedrella nodiflora; Fabaceae: Abrus precatorius, Canavalia gladiata, Crotalaria retusa, Mimosa pigra, Senna occidentalis, Senna obtusifolia). Many of them were most likely introduced in the precolonial period (Binggeli et al. 1998). However, species with low numbers of specimens could still be naturalized only locally. Field observations are urgently needed to ascertain the status (casual/naturalized/invasive) of most species in our database.
Interestingly, height had a significant negative effect on the number of records in Fabaceae. Small shrubs and forbs reach reproductive maturity earlier than tall trees and could therefore have shorter generation time and potentially higher rates of population increase.

CONCLUSION
The comparison of alien Asteraceae and Fabaceae in the flora of D.R. Congo has highlighted contrasting patterns in terms of geographic origin, life form, introduction pathways, occurrence, and trait divergence with the native flora. The striking discrepancies between the two families call for analyses of alien introductions at the family level and warn against generalisations based on the global flora.
Information on alien plant species in D.R. Congo is scanty. Future research should be directed in at least two directions. First, producing a reliable checklist validated by critical examination of herbarium material is a key priority. Secondly, fieldwork is urgently needed to assess the status of alien species, and possible impacts on native biota across the different natural regions of D.R. Congo. This is indeed a huge task, considering the size of the country and logistic difficulties. Protected areas should be priority targets in this process (Foxcroft et al. 2013), especially in parts of the country subjected to increasing anthropogenic pressure.