Seed consumption by small fish follows peak seed availability in a tropical dry forest river

Seed consumption and dispersal by fish has been more extensively described in natural Neotropical large river systems of Amazonia, where ichthyochory follows a seasonal gradient associated with a floodpulse that creates long-lasting seasonally flood zones. It has been shown that it is relevant in maintaining the plant community structure of wetlands, but its effects on plant communities in seasonal dry forests are largely unknown. The Amacuzac hydrological system, which runs through one of the most extensive seasonal tropical dry forests of Central Mexico, shows a marked climatic seasonality, which in turn determines a transient increase in river flow and its potential to overflow, resulting in a drag effect on banks, and in the river carrying three times more seeds in the rainy than in the dry season. Another characteristic of the system is the coexistence of native and non-native fish species, due to the fact that the middle part of the river receives discharges from four tributaries that cross important urban, industrial, and agricultural districts as well as an extensive network of ornamental fish farms. Therefore, we hypothesize that rates of seed consumption by fish would correspondingly increase during rainy seasons, and that both native and non-native fish would consume seeds in similar proportions. We evaluated seed consumption by fish by (a) identifying and estimating the frequency and number of fish species whose stomachs contained seeds, and determining the percentage of each fishes’ diet corresponding to seeds with respect to total food items, and (b) by evaluating if there were seasonal differences in the consumption of intact seeds by fish and between native vs. non-native species. We found two native species - Astyanax aeneus and Notropis moralesi, and two non-native species - Aequidens rivulatus and Amatitlania nigrofasciata containing seeds in their stomachs. The number of individuals with seeds was significantly higher in the rainy season than in the dry season, with a higher proportion of non-native fishes carrying seeds, but only in the rainy season. We found significant differences between the fish species in the proportion of seeds consumed. Fishes follow the peak of seed availability after a flood pulse released them from the soil seed banks. Thus, fishes consume fruits/seeds on a seasonal basis, however in this case it is explained by a different mechanism other than the timing of seed production by trees. As with hydrochory in tropical dry forests, seed consumption/dispersal by fish inhabiting rivers within this ecosystem has been overlooked in the ecology and conservation literature. This evidence is relevant for tropical dry forest ecosystem dynamics and management strategies in riparian corridors.

Methods
During 2015, we performed an intensive monthly sampling of fish in six locations along the Amacuzac River. Sampling was carried out in 200 m of river stretches, covering different types of habitats (rapids, parallel pools, backwaters and canals). The sampling effort was one hour. Fish were collected using cast nets of 2 m diameter and 20 mm mesh, and were kept cold for later analysis in the laboratory. We randomly selected 10-15 fish specimens per site from each species collected. Fish were dissected ventrally, leaving the visceral cavity exposed for extraction and for contents analysis by means of a stereoscopic microscope (Nikon SMZ-2T). Specimens were deposited in the fish collection (CICIB) at the Autonomous University of the State of Morelos (UAEM). The taxonomic identification of species was based on the criteria shown in Table S1, and with the help of the specialized staff at CICIB-UAEM. We determined the proportion of seeds with respect to total food items observed in stomachs using the numerical grid method (Lagler, 1956; Windell & Bowen, 1978).

Once we knew which fish species had seeds in their stomachs, we did a second field survey during the dry and rainy season of 2016, collecting only the four species identified in 2015 (Table S2). The sampling protocol and objectives were similar to that of the previous year, and were based on the methodology used by Mejía-Mojica et al. (2012). We sampled five locations in the same area of previous years’ sampling (Figure 1). Between 15-30 specimens were taken per species and location. Seeds were identified to the genus level and classified according to known original dispersal syndromes (Table S3 and S4).

鱼类对种子的取食与传播，在亚马逊流域的新热带大型自然河流水系中已有较为充分的研究。其中鱼媒传播（ichthyochory）呈现出与洪水脉冲相关的季节性梯度，该洪水脉冲会形成长期存在的季节性洪泛区。已有研究表明，鱼媒传播对维持湿地植物群落结构具有重要意义，但其对季节性热带旱林植物群落的影响仍鲜为人知。
阿马库萨克（Amacuzac）水文系统流经墨西哥中部规模最大的季节性热带旱林之一，其气候季节特征显著，这直接导致河流流量短暂升高并具备漫溢潜力，进而对河岸产生冲刷作用，且雨季河流携带的种子数量较旱季高出三倍。该水系的另一特征是本土与外来鱼类物种共存——河流中段承接四条支流的来水，这些支流流经重要的城市、工业与农业区域，同时串联起大规模的观赏鱼养殖网络。
据此，本研究提出两项假说：其一，鱼类取食种子的频次将在雨季显著升高；其二，本土与外来鱼类取食种子的比例相近。本研究通过以下两方面评估鱼类的种子取食行为：（a）鉴定并统计胃内含种子的鱼类物种的出现频率与数量，并计算各物种食谱中种子占总食物的百分比；（b）分析鱼类对完整种子的取食是否存在季节差异，以及本土与外来鱼类之间的取食差异。
本研究发现两种本土鱼类——Astyanax aeneus与Notropis moralesi，以及两种外来鱼类——Aequidens rivulatus与Amatitlania nigrofasciata的胃内含有种子。雨季时胃内含种子的鱼类个体数显著高于旱季，且仅在雨季，外来鱼类携带种子的比例更高。不同鱼类物种的种子取食比例存在显著差异。洪水脉冲将土壤种子库中的种子释放后，鱼类会追踪种子可获得性的峰值。因此，鱼类的果实/种子取食具有季节性，但本研究中的这一规律并非由树木的种子产季所决定，而是由另一套机制调控。与水媒传播（hydrochory）在热带旱林的研究现状类似，栖息于该生态系统河流中的鱼类对种子的取食与传播，在生态学与保护生物学文献中长期被忽视。本研究的相关发现对热带旱林生态系统动态及河岸带的管理策略具有重要参考价值。

## 研究方法
2015年，研究团队对阿马库萨克河沿岸的6个采样点开展了月度密集采样。采样区域为200米长的河段落，涵盖急流、平行池、回水区与运河等多种生境类型，单次采样时长为1小时。研究采用直径2米、网目20毫米的抛网采集鱼类样本，并将样本低温保存以用于后续实验室分析。每个采样点的采集物种中，随机选取10~15尾个体进行解剖。通过腹侧解剖暴露体腔，提取胃容物并借助体视显微镜（尼康SMZ-2T）开展内容物分析。标本被保存于莫雷洛斯州自治大学（UAEM）的鱼类标本馆（CICIB）。物种分类鉴定依据表S1中的标准，并得到CICIB-UAEM专业人员的协助。本研究采用数值网格法（Lagler, 1956; Windell & Bowen, 1978）计算胃容物中种子占总食物的比例。

在明确胃内含种子的鱼类物种后，研究团队于2016年的旱季与雨季开展了第二次野外调查，仅采集2015年已鉴定出的4个物种（表S2）。本次采样的方案与目标与上年一致，参考了Mejía-Mojica等人（2012）的研究方法。采样点设置于往年采样的同一区域内的5个点位（图1），每个物种在每个采样点采集15~30尾个体。研究将种子鉴定至属级水平，并依据已知的原始传播综合征进行分类（表S3与S4）。